Translocation and Stability of Replicative DNA Helicases upon Encountering DNA-Protein Cross-links

DNA-protein cross-links (DPCs) are formed when cells are exposed to various DNA-damaging agents. Because DPCs are extremely large, steric hindrance conferred by DPCs is likely to affect many aspects of DNA transactions. In DNA replication, DPCs are first encountered by the replicative helicase that moves at the head of the replisome. However, little is known about how replicative helicases respond to covalently immobilized protein roadblocks. In the present study we elucidated the effect of DPCs on the DNA unwinding reaction of hexameric replicative helicases in vitro using defined DPC substrates. DPCs on the translocating strand but not on the nontranslocating strand impeded the progression of the helicases including the phage T7 gene 4 protein, simian virus 40 large T antigen, Escherichia coli DnaB protein, and human minichromosome maintenance Mcm467 subcomplex. The impediment varied with the size of the cross-linked proteins, with a threshold size for clearance of 5.0–14.1 kDa. These results indicate that the central channel of the dynamically translocating hexameric ring helicases can accommodate only small proteins and that all of the helicases tested use the steric exclusion mechanism to unwind duplex DNA. These results further suggest that DPCs on the translocating and nontranslocating strands constitute helicase and polymerase blocks, respectively. The helicases stalled by DPC had limited stability and dissociated from DNA with a half-life of 15–36 min. The implications of the results are discussed in relation to the distinct stabilities of replisomes that encounter tight but reversible DNA-protein complexes and irreversible DPC roadblocks.     

Effect of vegetation on density thresholds of adult desert locust gregarization from survey data in Mauritania

In the desert locust, Schistocerca gregaria (Forskål) (Orthoptera: Acrididae), the threshold density inducing the gregarization phenomenon has never been determined under natural conditions. The influence of environmental factors on this phenomenon has been studied mostly in controlled environments. Based on data collected during several years by the survey teams of the National Center for Locust Control in Mauritania, we analyzed the influence of locust density, vegetation cover, and vegetation status on the probability of observing gregarious locusts. We assumed that a probability to observe gregarious locusts of 0.5 corresponded to the density threshold of gregarization. The results showed in detail the change in the threshold of gregarization according to the cover and status of the vegetation. Low cover and dry vegetation led to a low density threshold of gregarization probably due to high probability of individuals to touch each other. Dense and green vegetation favored a high threshold of gregarization probably due to a dispersion of the individuals and a low probability of individual encounters. These findings should help the management of locusts and decision making during control operations.     

New bioactive dihydrofuranocoumarins from the roots of the Tunisian Ferula lutea (Poir.) Maire

A phytochemical investigation of the roots of Ferula lutea (Poir.) Maire led to the isolation of new dihydrofuranocoumarins as two inseparable isomers, (?)-5-hydroxyprantschimgin 1 and (?)-5-hydroxydeltoin 2, together with eight known compounds, (?)-prantschimgin 3, (?)-deltoin 4, psoralen 5, xanthotoxin 6, umbelliferone 7, caffeic acid 8, β-sitosterol 9 and stigmasterol 10. Their structures were elucidated on the basis of extensive spectroscopic methods, including 1D and 2D NMR experiments and mass spectroscopy analysis, as well as by comparison with literature data. The anti-acetylcholinesterase and cytotoxic effects of the isolates and antioxidant activities of the mixture (1+2) were also evaluated in this work. Results showed that the mixture (1+2) has the most cytotoxic activity with IC₅₀ values 0.29 ± 0.05 and 1.61 ± 0.57 μM against the cell lines HT-29 and HCT 116, respectively. The greatest acetylcholinesterase inhibitory activity (IC₅₀ = 0.76 ± 0.03) was exhibited by the xanthotoxin 6. In addition, the mixture (1+2) was investigated for its antioxidant activity and showed IC₅₀ values 18.56, 13.06, 7.59, and 4.81 μM towards DPPH free radical scavenging, ABTS radical monocation, singlet oxygen and hydrogen peroxide, respectively.     

Rosiglitazone Treatment of Type 2 Diabetic db/db Mice Attenuates Urinary Albumin and Angiotensin Converting Enzyme 2 Excretion

Alterations within the renal renin angiotensin system play a pivotal role in the development and progression of cardiovascular and renal disease. Angiotensin converting enzyme 2 (ACE2) is highly expressed in renal tubules and has been shown to be renoprotective in diabetes. The protease, a disintegrin and metalloprotease (ADAM) 17, is involved in the ectodomain shedding of several transmembrane proteins including ACE2. Renal ACE2 and ADAM17 were significantly increased in db/db mice compared to controls. We investigated the effect of the insulin sensitizer, rosiglitazone, on albuminuria, renal ADAM17 protein expression and ACE2 shedding in db/db diabetic mice. Rosiglitazone treatment of db/db mice normalized hyperglycemia, attenuated renal injury and decreased urinary ACE2 and renal ADAM17 protein expression. Urinary excreted ACE2 is enzymatically active. Western blot analysis of urinary ACE2 demonstrated two prominent immunoreactive bands at approximately 70 & 90 kDa. The predominant immunoreactive band is approximately 20 kDa shorter than the one demonstrated for kidney lysate, indicating possible ectodomain shedding of active renal ACE2 in the urine. Therefore, it is tempting to speculate that renoprotection of rosiglitazone could be partially mediated via downregulation of renal ADAM17 and ACE2 shedding. In addition, there was a positive correlation between blood glucose, urinary albumin, plasma glucagon, and triglyceride levels with urinary ACE2 excretion. In conclusion, urinary ACE2 could be used as a sensitive biomarker of diabetic nephropathy and for monitoring the effectiveness of renoprotective medication.     

Optimization of the Extraction Process by Response Surface Methodology of Protein Isolate from Defatted Jujube (Zizyphus lotus L.) Seeds

International Journal of Peptide Research and Therapeutics - In this study, response surface methodology, based on Box-Behnken design, was used to optimize the extraction conditions of protein...     

Characterization of human serum albumin's interactions with safranal and crocin using multi-spectroscopic and molecular docking techniques

Interaction mechanisms of human serum albumin (HSA) with safranal and crocin were studied using UV–Vis absorption, fluorescence quenching and circular dichroism (CD) spectroscopies as well as molecular docking techniques. Changes in absorbance and fluorescence of HSA upon interactions with both compounds were attributed to their binding to amino acid chromophores located in subdomains IIA and IIIA. Fluorescence secondary inner filter effect was excluded using 278 nm and 340 nm as the wavelengths of HSA's excitation and fluorescence while safranal and crocin absorbed at 320 nm and 445 nm, respectively. Stern-Volmer model revealed a static quenching mechanism involve the formation of non-fluorescent ground state complexes. Stern-Volmer, Hill, Benesi-Hilbrand and Scatchard models gave apparent binding constants ranged in 4.25 × 10³ - 2.15 × 10⁵ for safranal and 7.67 × 10³ - 4.23 × 10⁵ L mol^?1 for crocin. CD measurements indicated that 13 folds of safranal and crocin unfolded the α-helix structure of HSA by 7.47–21.20%. In-silico molecular docking revealed selective exothermic binding of safranal on eight binding sites with binding energies ranged in ?3.969 to ?6.6.913 kcal/mol. Crocin exothermally bound to a new large pocket located on subdomain IIA (sudlow 1) with binding energy of ?12.922 kcal/mol.These results confirmed the formation of HSA stable complexes with safranal and crocin and contributed to our understanding for their binding characteristics (affinities, sites, modes, forces … etc.) and structural changes upon interactions. They also proved that HSA can solubilize and transport both compounds in blood to target tissues. The results are of high importance in determining the pharmacological properties of the two phytochemical compounds and for their future developments as anticancer, antispasmodic, antidepressant or aphrodisiac therapeutic agents.