Progression of mycotoxin and nutrient concentrations in wheat after inoculation with Fusarium culmorum

The objective of this study was to follow the mycotoxin formation and changes in nutrient composition of wheat (cv. Ritmo) artificially inoculated with Fusarium culmorum. From anthesis until harvest, samples were taken once a week from the inoculated and control plots. The investigations were focused on monitoring the progression of the contamination of the wheat kernels with deoxynivalenol (DON) and zearalenone (ZON). Both the uncontaminated control kernels and the contaminated kernels were examined also for the presence of zearalenone-4-beta-D-glucopyranoside and several trichothecenes at harvest. Furthermore, the impact of the Fusarium inoculation on some nutrients as starch, crude protein, amino acid composition, crude ash, non starch polysaccharides (NSP) as well as viscosity and thousand seed weight (TSW) was examined. Also proteolytic and amylolytic activity as well as the NSP-degrading enzyme activities of inoculated and control samples were analysed at the time of harvest. DON was detected in higher concentrations and in earlier stages, while ZON was found later and in smaller amounts. On average 7.79?mg/kg DM of DON and 100?μg/kg DM of ZON were found in the inoculated kernels at the time of harvest. Neither in the contaminated nor in the control samples glucose conjugates of ZON (Zearalenone-4-beta-D-glucopyranoside) were detected. Moreover, the infection with Fusarium culmorum had pronounced effects on some quality parameters. The crude protein content of the inoculated kernels showed significantly higher values over the whole period compared to the control kernels. The protein content of the inoculated kernels amounted 13.9% DM at harvest, while only a concentration of 12.5% DM was detected in the control samples. Similarly, in almost all stages of development the crude ash content of inoculated samples was higher than in control samples. These distinct differences in kernel composition resulted possibly from the changes of the thousand seed weight. In the present work the grain harvested from the control plots showed a significantly higher TSW (24.2?g) as compared to their inoculated counterparts (15.5?g). Despite lower extract viscosity of inoculated samples at time of harvest, the content of soluble NSP of inoculated plots was higher than in control samples at the same time. Moreover, inoculation resulted in markedly increased activities of protease, amylase and several NSP-degrading enzyme activities. This would suggest that the cell wall penetrating properties of the fungus itself and/or that the fungus induced alterations of the metabolic activity of the embryo or other constituents of the wheat kernel could be responsible.     

Chemotaxonomic Screening of Seed Oils from the Family Saxifragaceae and Comparison with Data on Seed Oils from Grossulariaceae Obtained from Literature

Seeds of 25 members of the family Saxifragaceae, 1 × Astilbe, 1 × Darmera, 1 × Leptarrhena, 1 × Tellima, 3 × Mitella, and 18 × Saxifraga were investigated regarding oil content, as well as composition and content of fatty acids and vitamin E active compounds. The results were compared with results obtained from literature for members of the genus Ribes belonging to the closely related family Grossulariaceae to find chemometric differences between the different genera and between members of the family Saxifragaceae and Grossulariaceae, respectively. Members of the family Saxifragaceae are dominated by high amounts of linoleic and α‐linolenic acid which together account for about 80% of the total fatty acids. While α‐linolenic acid is characteristic for members of the genus Saxifraga, in other genera, linoleic acid is predominant. In comparison to members of the family Saxifragaceae members of the family Grossulariaceae also contain γ‐linolenic acid and stearidonic acid which allow a significant differentiation between both families. By principle component analysis, members of both families were divided into three distinct groups, i) species with a high content of α‐linolenic acid (genus Saxifraga), ii) species with high amounts of γ‐linolenic acid and stearidonic acid (genus Ribes), and iii) species with higher amounts of linoleic acid (other members of the family Saxifragaceae). The composition of the vitamin E active compounds was characterized by a high content of γ‐tocopherol in most members of the family Saxifragaceae, but no chemotaxonomic relevance.     

Structural and stability effects of phosphorylation: Localized structural changes in phenylalanine hydroxylase

Phosphorylation of phenylalanine hydroxylase (PAH) at Ser16 by cAMP-dependent protein kinase increases the basal activity of the enzyme and its resistance to tryptic proteolysis. The modeled structures of the full-length phosphorylated and unphosphorylated enzyme were subjected to molecular dynamics simulations, and we analyzed the energy of charge-charge interactions for individual ionizable residues in the final structures. These calculations showed that the conformational changes induced by incorporation of phosphate were localized and limited mostly to the region around the phosphoserine (Arg13-Asp17) and a region around the active site in the catalytic domain that includes residues involved in the binding of the iron and the substrate L-Phe (Arg270 and His285). The absence of a generalized conformational change was confirmed by differential scanning calorimetry, thermal-dependent circular dichroism, fluorescence spectroscopy, and limited chymotryptic proteolysis of the phosphorylated and unphosphorylated PAH. Our results explain the effect of phosphorylation of PAH on both the resistance to proteolysis specifically by trypsin-like enzymes and on the increase in catalytic efficiency.     

Progression of the mycotoxin and nutrient concentration in wheat after inoculation with<Emphasis Type="Italic">Fusarium culmorum</Emphasis>

The aim of the study was to follow the mycotoxin formation and the changes in nutrient composition of wheat artificially inoculated withFusarium cuimorum. From anthesis until harvest, samples were taken once a week from the inoculated and control plots. The investigations were focused on the progression of the contamination of the wheat with deoxyni-valenol (DON) and zearalenone (ZON). Furthermore, the impact of the Fusarium inoculation on the quality parameters starch, crude protein, crude ash, viscosity, non starch polysaccharides (NSP) and thousand seed weight was examined. Deoxynivalenol was detected in higher concentrations and in earlier stages, while zearalenone was found later and in smaller amounts. Moreover, the infection withFusarium cuimorum had pronounced effects on some quality parameters.     

L-phenylalanine binding and domain organization in human phenylalanine hydroxylase: a differential scanning calorimetry study

Human phenylalanine hydroxylase (hPAH) is a tetrameric enzyme that catalyzes the hydroxylation of L-phenylalanine (L-Phe) to L-tyrosine; a dysfunction of this enzyme causes phenylketonuria. Each subunit in hPAH contains an N-terminal regulatory domain (Ser2-Ser110), a catalytic domain (Asp112-Arg410), and an oligomerization domain (Ser411-Lys452) including dimerization and tetramerization motifs. Two partially overlapping transitions are seen in differential scanning calorimetry (DSC) thermograms for wild-type hPAH in 0.1 M Na-Hepes buffer, 0.1 M NaCl, pH 7.0. Although these transitions are irreversible, studies on their scan-rate dependence support that the equilibrium thermodynamics analysis is permissible in this case. Comparison with the DSC thermograms for truncated forms of the enzyme, studies on the protein and L-Phe concentration effects on the transitions, and structure-energetic calculations based on a modeled structure support that the thermal denaturation of hPAH occurs in three stages: (i) unfolding of the four regulatory domains, which is responsible for the low-temperature calorimetric transition; (ii) unfolding of two (out of the four) catalytic domains, which is responsible for the high-temperature transition; and (iii) irreversible protein denaturation, which is likely responsible for the observed exothermic distortion in the high-temperature side of the high-temperature transition. Stages 1 and 2 do not appear to be two-state processes. We present an approach to the analysis of ligand effects on DSC transition temperatures, which is based on the general binding polynomial formalism and is not restricted to two-state transitions. Application of this approach to the L-Phe effect on the DSC thermograms for hPAH suggests that (i) there are no binding sites for L-Phe in the regulatory domains; therefore, contrary to the common belief, the activation of PAH by L-Phe seems to be the result of its homotropic cooperative binding to the active sites. (ii) The regulatory domain appears to be involved in cooperativity through its interactions with the catalytic and oligomerization domains; thus, upon regulatory domain unfolding, the cooperativity in the binding of L-Phe to the catalytic domains seems to be lost and the value of the L-Phe concentration corresponding to half-saturation is increased. Overall, our results contribute to the understanding of the conformational stability and the substrate-induced cooperative activation of this important enzyme.     

Proliferation in murine epidermis after minor mechanical stimulation. Part 1. Sustained increase in keratinocyte production and migration

It was our objective to obtain an insight into the details and dynamics of the cell proliferative changes following minor barrier disruption, the mechanisms of recovery, and their regulation. Hair of the dorsal area of DBA2-mice was removed and the epidermis was tape stripped. Tritiated thymidine was injected into groups of mice at daily intervals thereafter. Labelling and nuclear densities were measured at several time intervals later in the various epidermal strata to characterize cell production and cell fluxes through the tissue. A dramatic proliferative response was observed at 24 h when the labelling density increased more than sixfold in the basal layer. Labelled cells rapidly appeared in suprabasal layers within a few hours in large quantities while this process took over 2 days in normal skin. Some cycling cells were also found in the suprabasal layer (pulse labelling at 24 h) in contrast with the controls. The cellular flux through the suprabasal layers was drastically (20-fold) increased and the transit time was shortened. Although the nuclear density in the basal layer showed only moderate changes it increased four-fold in the suprabasal layer within 5 days. A kinetic model analysis suggested that the cell cycle time of proliferative cells dropped from a normal value of about 200 h to less than 12 h post tape strip. After 7 days, the proliferative activation still persisted, even though at 3 days post tape strip the stratum corneum had been re-established. Hence, a mild mechanical alteration with removal of some parts of the cornified layer in mouse backskin epidermis triggers a huge proliferative response with massive overproduction of cells that lasts at least 7 days. Our findings suggest that the re-establishment of the cornified layer does not immediately shut down cell proliferation and that more complex, slower (long-term) regulatory processes are involved.     

Duplicated sequence motif in the long terminal repeat of maedi-visna virus extends cell tropism and is associated with neurovirulence

Maedi-visna virus (MVV) is a lentivirus of sheep causing chronic inflammatory disease of the lungs (maedi) and the nervous system (visna). We have previously shown that a duplicated sequence in the long terminal repeat (LTR) of MVV is a determinant of cell tropism. Here, we demonstrate that deletion of a CAAAT sequence from either one of the repeats resulted in poor virus growth in sheep choroid plexus cells. A duplication in the LTR encompassing the CAAAT sequence was found in four neurological field cases that were sequenced, but no duplication was present in the LTRs from seven maedi cases; one maedi isolate was mixed. These results indicate that the duplication in the LTR is associated with neurovirulence.     

The Y deletion gr/gr and susceptibility to testicular germ cell tumor

Testicular germ cell tumor (TGCT) is the most common cancer in young men. Despite a considerable familial component to TGCT risk, no genetic change that confers increased risk has been substantiated to date. The human Y chromosome carries a number of genes specifically involved in male germ cell development, and deletion of the AZFc region at Yq11 is the most common known genetic cause of infertility. Recently, a 1.6-Mb deletion of the Y chromosome that removes part of the AZFc region—known as the “gr/gr” deletion—has been associated with infertility. In epidemiological studies, male infertility has shown an association with TGCT that is out of proportion with what can be explained by tumor effects. Thus, we hypothesized that the gr/gr deletion may be associated with TGCT. Using logistic modeling, we analyzed this deletion in a large series of TGCT cases with and without a family history of TGCT. The gr/gr deletion was present in 3.0% (13/431) of TGCT cases with a family history, 2% (28/1,376) of TGCT cases without a family history, and 1.3% (33/2,599) of unaffected males. Presence of the gr/gr deletion was associated with a twofold increased risk of TGCT (adjusted odds ratio [aOR] 2.1; 95% confidence interval [CI] 1.3–3.6; P = .005) and a threefold increased risk of TGCT among patients with a positive family history (aOR 3.2; 95% CI 1.5–6.7; P = .0027). The gr/gr deletion was more strongly associated with seminoma (aOR 3.0; 95% CI 1.6–5.4; P = .0004) than with nonseminoma TGCT (aOR 1.5; 95% CI 0.72–3.0; P = .29). These data indicate that the Y microdeletion gr/gr is a rare, low-penetrance allele that confers susceptibility to TGCT.     

Infrared microspectroscopy of individual human cervical cancer (HeLa) cells suspended in growth medium

We report for the first time the infrared spectra of individual human cervical cancer (HeLa) cells suspended in buffer or cell culture medium. Although we did not establish whether these cells were viable at the time of spectral data acquisition, we believe that the methodology used is applicable to the study of live cells. Data were collected either from entire cells, using 25- to 40-microm apertures, or via an imaging approach, where pixels measuring 6.25 x 6.25 microm were assembled to form a map of a cell in suspension. Measurements were carried out both in reflection/absorption and in transmission modes. The results reported here might have far-reaching implications for the use of infrared microspectroscopy to monitor cell proliferation, drug response, and other cell biological parameters in live cells.