The concerted action of a positive charge and hydrogen bonds dynamically regulates the pKa of the nucleophilic cysteine in the NrdH‐redoxin family

NrdH‐redoxins shuffle electrons from the NADPH pool in the cell to Class Ib ribonucleotide reductases, which in turn provide the precursors for DNA replication and repair. NrdH‐redoxins have a CVQC active site motif and belong to the thioredoxin‐fold protein family. As for other thioredoxin‐fold proteins, the pK_a of the nucleophilic cysteine of NrdH‐redoxins is of particular interest since it affects the catalytic reaction rate of the enzymes. Recently, the pK_a value of this cysteine in Corynebacterium glutamicum and Mycobacterium tuberculosis NrdH‐redoxins were determined, but structural insights explaining the relatively low pK_a remained elusive. We subjected C. glutamicum NrdH‐redoxin to an extensive molecular dynamics simulation to expose the factors regulating the pK_a of the nucleophilic cysteine. We found that the nucleophilic cysteine receives three hydrogen bonds from residues within the CVQC active site motif. Additionally, a fourth hydrogen bond with a lysine located N‐terminal of the active site further lowers the cysteine pK_a. However, site‐directed mutagenesis data show that the major contribution to the lowering of the cysteine pK_a comes from the positive charge of the lysine and not from the additional Lys‐Cys hydrogen bond. In 12% of the NrdH‐redoxin family, this lysine is replaced by an arginine that also lowers the cysteine pK_a. All together, the four hydrogen bonds and the electrostatic effect of a lysine or an arginine located N‐terminally of the active site dynamically regulate the pK_a of the nucleophilic cysteine in NrdH‐redoxins.     

Modeling of polyphenols extraction from pomegranate by-product using rotatable central composite design of experiments

There is a growing request to find an effective method of polyphenols extraction from agro-industry by-product as pomegranate. In this study, response surface methodology (RSM) was used to explore the effect of three factors on ultrasonic assisted extraction (UAE) of total polyphenols (TP), total flavonoids (TF) and condensed tannins (CT) from pomegranate peels. The optimal conditions were determined for each phenolic compound using regression model equations and 3-D plots. The high TP, TF and CT content were obtained with, respectively, liquid/solid ratio of 20.00, 9.77, 9.77, extraction time of 36.38, 41.82, 30.39 min and 36.00, 83.64, 59.26% of ethanol percentage. In fact, liquid/solid ratio of 20, extraction time of 30.94 min and 59.26% of ethanol gives the highest contents of TP, TF and CT simultaneously. The experimental values using optimal conditions agreed with the predicted values. The pomegranate peels extract obtained under optimum conditions have an effective antioxidant activity as determined by ABTS and DPPH assays.     

Proteolytic activities in Phaseolus vulgaris cotyledons under copper stress

The changes in the protease activities of bean cotyledons were investigated in response to copper stress. Assays using synthetic substrates and specific protease inhibitors followed by activity measurements and electrophoresis analysis allowed to study the classes of enzymes involved in the storage protein mobilization during the germination of bean (Phaseolus vulgaris L) seeds, and then identify which ones were affected in the presence of 200 μM CuCl₂ in the imbibition medium. Copper treatment affected embryo growth and total protease activity. The results of SDS-gelatin-PAGE show that Cu excess led to a decrease in protease activity of 45 to 66 kDa. Moreover, cysteine-, aspartic- and metallo-protease activities were markedly lowered under copper stress, while serine-protease one was enhanced as well as its activity dependent abundance in comparison with control. However, the relative distribution of major cysteine protease in H₂O-germinated seeds was significantly diminished after Cu exposure. Thus, copper excess can disturb the nitrogen freeing from reserve tissues at enzymatic level; differential responses of protease classes are discussed, notably, cysteine protease in the way of storage protein mobilization and serine protease in protective mechanism one.     

Assessment of Morphine-induced Hyperalgesia and Analgesic Tolerance in Mice Using Thermal and Mechanical Nociceptive Modalities

Opioid-induced hyperalgesia and tolerance severely impact the clinical efficacy of opiates as pain relievers in animals and humans. The molecular mechanisms underlying both phenomena are not well understood and their elucidation should benefit from the study of animal models and from the design of appropriate experimental protocols. We describe here a methodological approach for inducing, recording and quantifying morphine-induced hyperalgesia as well as for evidencing analgesic tolerance, using the tail-immersion and tail pressure tests in wild-type mice. As shown in the video, the protocol is divided into five sequential steps. Handling and habituation phases allow a safe determination of the basal nociceptive response of the animals. Chronic morphine administration induces significant hyperalgesia as shown by an increase in both thermal and mechanical sensitivity, whereas the comparison of analgesia time-courses after acute or repeated morphine treatment clearly indicates the development of tolerance manifested by a decline in analgesic response amplitude. This protocol may be similarly adapted to genetically modified mice in order to evaluate the role of individual genes in the modulation of nociception and morphine analgesia. It also provides a model system to investigate the effectiveness of potential therapeutic agents to improve opiate analgesic efficacy.     

Responses of Proteolytic Enzymes in Embryonic Axes of Germinating Bean Seeds under Copper Stress

The changes in protease activities in embryonic axes during the first days of bean (Phaseolus vulgaris L.) seed germination were investigated in response to copper stress. Synthetic substrates and specific protease inhibitors have been used to define qualitatively and quantitatively different catalytic classes, particularly endoproteases (EP), carboxypeptidases (CP) and aminopeptidases (AP), then identify which ones were affected in the presence of copper. In fact, a failure in storage proteins mobilization and a disorder of nitrogen supply at enzymatic level occurred in Cu. In fact, Cu inhibited azocaseinolytic activity (ACA) and cysteine-, aspartic-, serine-, and metallo-endopeptidases activities (Cys-EP, Asp-EP, Ser-Ep, and Met-EP, respectively). Besides, Cu affected leucine- and proline-aminopeptidases (LAP and PAP, respectively) and glycine-carboxypeptidases (Gly-CP). The proteolytic responses might also be associated with the decrease in defense capacity in the Cu-treated embryos.     

Biological effects of alkaloïds extracted from three plants of Moroccan arid areas on the desert locust

Abstract. The effects of three plant species, Calotropis procera , Zygophyllum gaetulum and Peganum harmala on survival, feeding behaviour and reproduction in the desert locust ( Schistocerca gregaria ) were studied under laboratory conditions. The species originate in Moroccan arid areas and were seen to be avoided by locusts during the 1995 upsurge in Morocco. Alkaloids were extracted from leaves of the plants and applied topically to lettuce for leaves which were then offered with no other food to young adults of the desert locust for 3 days. The locust were then fed on untreated lettuce for 15 days. Controls were fed with lettuce leaves treated with ethanol. The results indicate that the alkaloids extracted from each of the three plant species cause a significant mortality compared to untreated controls and reduced food intake, as well as causing weight loss. Alkaloids extracted from C. procera and Z. gaetulum prevent sexual maturity both in males and females. However, alkaloids extracted from P. harmala merely delay sexual maturity by at least 8 days and engender a reduction in female fecundity and hatching rate compared to untreated controls. The presence and the concentration of alkaloids and other secondary substances in the investigated plants may explain the avoidance behaviour manifested by locusts under natural conditions.     

Evolutionary Trade-Offs Underlie the Multi-faceted Virulence of Staphylococcus aureus

Bacterial virulence is a multifaceted trait where the interactions between pathogen and host factors affect the severity and outcome of the infection. Toxin secretion is central to the biology of many bacterial pathogens and is widely accepted as playing a crucial role in disease pathology. To understand the relationship between toxicity and bacterial virulence in greater depth, we studied two sequenced collections of the major human pathogen Staphylococcus aureus and found an unexpected inverse correlation between bacterial toxicity and disease severity. By applying a functional genomics approach, we identified several novel toxicity-affecting loci responsible for the wide range in toxic phenotypes observed within these collections. To understand the apparent higher propensity of low toxicity isolates to cause bacteraemia, we performed several functional assays, and our findings suggest that within-host fitness differences between high- and low-toxicity isolates in human serum is a contributing factor. As invasive infections, such as bacteraemia, limit the opportunities for onward transmission, highly toxic strains could gain an additional between-host fitness advantage, potentially contributing to the maintenance of toxicity at the population level. Our results clearly demonstrate how evolutionary trade-offs between toxicity, relative fitness, and transmissibility are critical for understanding the multifaceted nature of bacterial virulence.