The potential of plant bioactives for the prevention and therapy of diabetes is increasingly being recognized. In the present study we investigated the antidiabetic properties of an aqueous Bistorta ...officinalis Delarbre extract (BODE) by employing both in-vitro assays and in-vivo models. Multiple targets in glucose homeostasis which are involved in the regulation of the blood glucose level were affected by BODE in-vitro. The extract exhibited inhibitory activities towards the intestinal carbohydrate-hydrolysing enzymes α-amylase and α-glucosidase with IC
values of 81.5 μg/mL and 8.4 μg/mL, respectively. Furthermore, moderate reduction of the dipeptidyl peptidase-4 (DPP4) enzyme activity was evident when tested in the presence of 1.0 mg/mL BODE. A significant inhibition of the intestinal glucose transporter sodium-dependent glucose transporter 1 (SGLT1) in response to 1.0 mg/mL BODE was shown for Caco-2 cells mounted in Ussing chambers. High performance liquid chromatography-mass spectrometry analyses of the BODE revealed several plant bioactives including gallotannins, catechins and chlorogenic acid. Although our in-vitro data were promising, BODE-supplementation in the model organism Drosophila melanogaster lacked to confirm the antidiabetic effect of the extract in-vivo. Moreover, BODE failed to reduce blood glucose levels in chicken embryos (in-ovo). Hence, BODE is probably not a suitable candidate for developing a pharmaceutical against diabetes mellitus.
Heat stress is detrimental to food-producing animals and animal productivity remains suboptimal despite the use of heat abatement strategies during summer. Global warming and the increase of ...frequency and intensity of heatwaves are likely to continue and, thus, exacerbate the problem of heat stress. Heat stress leads to the impairment of physiological and cellular functions of ectothermic and endothermic animals. Therefore, it is critical to conceive ways of protecting animals against the pathological effects of heat stress. In experiments with endothermic animals highly sensitive to heat (Bos taurus), we have previously reported that heat-induced systemic inflammation can be ameliorated in part by nutritional interventions. The experiments conducted in this report described molecular and physiological adaptations to heat stress using Drosophila melanogaster and dairy cow models. In this report, we expand previous work by first demonstrating that the addition of a postbiotic from Aspergillus oryzae (AO) into the culture medium of ectothermic animals (Drosophila melanogaster) improved survival to heat stress from 30 to 58%. This response was associated with downregulation of genes involved in the modulation of oxidative stress and immunity, most notably metallothionein B, C, and D. In line with these results, we subsequently showed that the supplementation with the AO postbiotic to lactating dairy cows experiencing heat stress decreased plasma concentrations of serum amyloid A and lipopolysaccharide-binding protein, and the expression of interleukin-6 in white blood cells. These alterations were paralleled by increased synthesis of energy-corrected milk and milk components, suggesting enhanced nutrient partitioning to lactogenesis and increased metabolic efficiency. In summary, this work provides evidence that a postbiotic from AO enhances thermal tolerance likely through a mechanism that entails reduced inflammation.
During the erythrocytic cycle, Plasmodium falciparum is highly dependent on an adequate thiol status for its survival. Glutathione reductase as well as de novo synthesis of GSH are responsible for ...the maintenance of the intracellular GSH level. The first and rate-limiting step of the synthetic pathway is catalysed by gamma-glutamylcysteine synthetase (gamma-GCS). Using L-buthionine-(S, R)-sulphoximine (BSO), a specific inhibitor of the gamma-GCS, we show that the infection with P. falciparum causes drastic changes in the GSH metabolism of red blood cells (RBCs). Infected RBCs lose GSH at a rate 40-fold higher than non-infected RBCs. The de novo synthesis of the tripeptide was found to be essential for parasite survival. GSH depletion by BSO inhibits the development of P. falciparum with an IC(50) of 73 microM. The effect of the drug is abolished by supplementation with GSH or GSH monoethyl ester. Our studies demonstrate that the plasmodicidal effect of the inhibitor BSO does not depend on its specificity towards its target enzyme in the parasite, but on the changed physiological needs for the metabolite GSH in the P. falciparum-infected RBCs. Therefore the depletion of GSH is proposed as a chemotherapeutic strategy for malaria, and gamma-GCS is proposed as a potential drug target.
The polyamines putrescine, spermidine and spermine play an essential role in cell differentiation and proliferation. Inhibition of the rate-limiting enzymes of polyamine biosynthesis, ornithine ...decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC), has been proposed as a therapeutic strategy against cancer and parasitic infections. In the case of Plasmodium falciparum, the causative agent of malaria tropica, this approach is especially interesting, because here both key enzymes, ODC and AdoMetDC, are combined in a bifunctional protein, ODC/AdoMetDC. This arrangement has not been found in any other organism investigated so far. We report the cloning and recombinant expression of the ODC domain of P. falciparum in Escherichia coli. First, we expressed the mere recombinant ODC domain (rPfODC). Secondly, we expressed the recombinant ODC domain in conjunction with the preceding part of the hinge region of the bifunctional ODC/AdoMetDC (rPfHinge-ODC). K(m) values for L-ornithine were 47.3 microM for the rPfHinge-ODC and 161. 5 microM for the rPfODC. Both recombinant enzymes were inhibited by putrescine, but the K(i) value for the rPfHinge-ODC was 50.4 microM (IC(50)=157 microM), whereas the IC(50) for the rPfODC was 500 microM. Spermidine was a weak inhibitor in both cases. alpha-Difluoromethylornithine inhibited the rPfHinge-ODC with a K(i) value of 87.6 microM. For two novel ODC inhibitors, CGP52622A and CGP54619A, the K(i) values of the rPfHinge-ODC were in the nanomolar range.
Thialysine
N
ε-acetyltransferases and spermidine/spermine
N-acetyltransferases (SSAT) are closely related members of the GCN5-related
N-acetyltransferase superfamily. Accordingly, a putative ...orthologue from the human protozoan parasite
Leishmania major exhibits an almost equal similarity to human SSAT and thialysine
N
ε-acetyltransferase. Characterisation of the recombinantly expressed
L. major protein indicated that it represents a thialysine
N
ε-acetyltransferase, preferring thialysine (
S-aminoethyl-
l-cysteine) and structurally related amino acids as acceptor molecules. The known thialysine
N
ε-acetyltransferases contain five conserved amino acid residues that are replaced in SSAT sequences. Kinetic analyses of the respective recombinant mutant proteins suggest that Ser
82 and Thr
83 of
L. major thialysine
N
ε-acetyltransferase are key residues for acceptor binding. In addition, the conserved Leu
130 is tentatively involved in specific interaction with the sulphur-containing side chain of thialysine. The presence of these three amino acid residues is suggested to be a means by which thialysine
N
ε-acetyltransferases can be distinguished from SSAT sequences.
Ethanolic and aqueous extracts of selected medicinal plants from Cameroon and Ghana were assessed for their in vitro anthelmintic activity by using the bovine filarial parasite Onchocerca ochengi and ...the free living nematode Caenorhabditis elegans, a model organism for research on nematode parasites. Worms were incubated in the presence of different concentrations of extracts and inhibitory effects were monitored at different time points. Among the extracts used in this study, ethanolic extracts of Anogeissus leiocarpus, Khaya senegalensis, Euphorbia hirta and aqueous extracts from Annona senegalensis and Parquetina nigrescens affected the growth and survival of C. elegans and O. ochengi significantly. The mortality was concentration dependent with an LC50 ranging between 0.38 and 4.00 mg/ml for C. elegans (after 72 h) and between 0.08 and 0.55 mg/ml for O. ochengi after a 24 h incubation time. Preliminary phytochemical screenings on these extracts revealed the presence of flavonoids, alkaloids, saponins, carbohydrates and tannins in the extracts. Accordingly, application of A. leiocarpus, K. senegalensis, E. hirta and A. senegalensis extracts could provide alternatives in the control of helminthic infections.
Plasmodium falciparum is the causative agent of malaria tropica. Due to the increasing resistance towards the commonly used plasmodicidal drugs there is an urgent need to identify and assess new ...targets for the chemotherapeutic intervention of parasite development in the human host. It is established that P. falciparum-infected erythrocytes are vulnerable to oxidative stress, and therefore efficient antioxidative systems are required to ensure parasite development within the host cell. The thioredoxin and glutathione redox systems represent two powerful means to detoxify reactive oxygen species and this article summarizes some of the recent work which has led to a better understanding of these systems in the parasite and will help to assess them as potential targets for the development of new chemotherapeutics of malaria.
The tripeptide glutathione plays a pivotal role in the maintenance of the thiol redox state of the cell and for the detoxification of reactive oxygen species. Glutathione is synthesized in two ...consecutive reactions by
γ-glutamylcysteine synthetase (
γ-GCS) and glutathione synthetase, respectively. The former enzyme represents the rate limiting step of the synthetic pathway. We have cloned the cDNA and gene of a putative
γ-GCS from
Plasmodium falciparum. The contiguous cDNA sequences obtained from various cDNA libraries of
P. falciparum K1 and 3D7 encompass 4206 bp or 4038 bp and encode polypeptides of 1119 and 1063 amino acids, respectively. The deduced amino acid sequences show four regions of homology (identity: 31.3–43.9%) to human and
Trypanosoma brucei
γ-GCS. These regions are interrupted by three large insertions between 94 and 239 amino acids. Within the first insert a variable repetitive motif was identified, which is responsible for the differing sizes of the sequences. We have analysed this phenomenon in five additional
P. falciparum strains and found a high degree of variability in the number of the repeated octamer (Y/C)S(N/D)LQQ(Q/R). Therefore the predicted molecular mass of the proteins from different
P. falciparum strains ranges from 124.4 to 133.2 kDa, which is almost twice that of the catalytic subunit of the human host enzyme. Isolation of three genomic clones revealed that the gene does not contain introns.
P. falciparum
γ-GCS transcription peaks in trophozoites (24–30 h) suggesting that the antioxidant glutathione is predominantly produced at a time where hemoglobin degradation and the simultaneous formation of reactive oxygen species is maximal.
S-Adenosylmethionine decarboxylase (AdoMetDC) is a key enzyme of the polyamine synthetic pathway providing decarboxylated S-adenosylmethionine for the formation of spermidine and spermine, ...respectively. The catalytic activity of the AdoMetDC from the free-living nematode Caenorhabditis elegans highly depends on the presence of an activator molecule. Putrescine, a well-known stimulator of mammalian AdoMetDC activity, enhances the catalytic activity of the nematode enzyme 350-fold. Putrescine stimulation is discussed as a regulatory mechanism to relate putrescine abundance with the synthesis of spermidine and spermine. In contrast to any other known AdoMetDC, spermidine and spermine also represent significant activators of the nematode enzyme. However, the biological significance of the observed stimulation by these higher polyamines is unclear. Although C. elegans AdoMetDC exhibits a low specificity toward activator molecules, the amino acid residues that were shown to be involved in putrescine binding of the human enzyme are conserved in the nematode enzyme. Exchanging these residues by site-directed mutagenesis indicates that at least three residues, Thr192, Glu194 and Glu274, most likely contribute to activator binding in the C. elegans AdoMetDC. Interestingly, the mutant Glu194Gln exhibits a 100-fold enhanced basal activity in the absence of any stimulator, suggesting that this mutant protein mimics the conformational change usually induced by activator molecules. Furthermore, site-directed mutagenesis revealed that at least Glu33, Ser83, Arg91 and Lys95 are involved in posttranslational processing of C. elegans AdoMetDC.
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