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.     

Granzyme B-induced cell death involves induction of p53 tumor suppressor gene and its activation in tumor target cells

In this study we investigated the involvement of p53 in cytotoxic T-lymphocyte (CTL)-induced tumor target cell killing mediated by the perforin/granzymes pathway. For this purpose we used a human CTL clone (LT12) that kills its autologous melanoma target cells (T1), harboring a wild type p53. We demonstrated initially that LT12 kills its T1 target in a perforin/granzymes-dependent manner. Confocal microscopy and Western blot analysis indicated that conjugate formed between LT12 and T1 resulted in rapid cytoplasmic accumulation of p53 and its activation in T1 target cells. Cytotoxic assay using recombinant granzyme B (GrB) showed that this serine protease is the predominant factor inducing such accumulation. Furthermore, RNA interference-mediated lowering of the p53 protein in T1 cells or pifithrin-alpha-induced p53-specific inhibition activity significantly decreased CTL-induced target killing mediated by CTL or recombinant GrB. This emphasizes that p53 is an important determinant in granzyme B-induced apoptosis. Our data show furthermore that when T1 cells were treated with streptolysin-O/granzyme B, specific phosphorylation of p53 at Ser-15 and Ser-37 residues was observed subsequent to the activation of the stress kinases ataxia telangiectasia mutated (ATM) and p38K. Treatment of T1 cells with pifithrin-alpha resulted in inhibition of p53 phosphorylation at these residues and in a significant decrease in GrB-induced apoptotic T1 cell death. Furthermore, small interference RNAs targeting p53 was also accompanied by an inhibition of streptolysin-O/granzyme B-induced apoptotic T1 cell death. The present study supports p53 induction after CTL-induced stress in target cells. These findings provide new insight into a potential role of p53 as a component involved in the dynamic regulation of the major pathway of CTL-mediated cell death and may have therapeutic implications.     

Isolation and characterisation of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) degrading actinomycetes and purification of PHBV depolymerase from newly isolatedStreptoverticillium kashmirense AF1

Streptoverticillium kashmirense AF1 with the ability to degrade a natural polymer, poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) was isolated from municipal sewage sludge by soil burial technique. The PHBV film was degraded by the action of extracellular enzymes secreted by the microorganisms. Degradation of PHBV was evident by the formation of clear zones of hydrolysis on the polymer containing mineral salt agar plates. The extent of PHBV degradation increased up to 30 days of incubation. Maximum production of PHBV depolymerase was observed both at pH 8 and pH 7, 45 °C, 1% substrate concentration and in the presence of lactose as an additional carbon source. Two types of extracellular PHBV depolymerases were purified fromS. kashmirense AF1 by gel permeation chromatography using Sephadex G-75. The molecular weights of the two proteins were found to be 35 and 45 kDa approximately, as determined by SDS-PAGE. The results of the Sturm test also showed more CO₂ production as a result of PHBV degradation, in the test as compared to control. The present findings indicated the degradation capabilities ofS. kashmirense AF1.     

Unique roles of aminophospholipid translocase Drs2p in governing efflux pump activity,ergosterol level,virulence traits,and host–pathogen interaction in Candida albicans

International Microbiology - Infections caused by Candida albicans are rising due to increment in drug resistance and a limited arsenal of conventional antifungal drugs. Thus, elucidating the novel...     

Galectin-3 exacerbates ox-LDL-mediated endothelial injury by inducing inflammation via integrin β1-RhoA-JNK signaling activation

Oxidized low-density lipoprotein (Ox-LDL)-induced endothelial cell injury plays a crucial role in the pathogenesis of atherosclerosis (AS). Plasma galectin-3 (Gal-3) is elevated inside and drives diverse systemic inflammatory disorders, including cardiovascular diseases. However, the exact role of Gal-3 in ox-LDL-mediated endothelial injury remains unclear. This study explores the effects of Gal-3 on ox-LDL-induced endothelial dysfunction and the underlying molecular mechanisms. In this study, Gal-3, integrin β1, and GTP-RhoA in the blood and plaques of AS patients were examined by ELISA and western blot respectively. Their levels were found to be obviously upregulated compared with non-AS control group. CCK8 assay and flow cytometry analysis showed that Gal-3 significantly decreased cell viability and promoted apoptosis in ox-LDL-treated human umbilical vascular endothelial cells (HUVECs). The upregulation of integrinβ1, GTP-RhoA, p-JNK, p-p65, p-IKKα, and p-IKKβ induced by ox-LDL was further enhanced by treatment with Gal-3. Pretreatment with Gal-3 increased expression of inflammatory factors (interleukin [IL]-6, IL-8, and IL-1β), chemokines(CXCL-1 and CCL-2) and adhesion molecules (VCAM-1 and ICAM-1). Furthermore, the promotional effects of Gal-3 on NF-κB activation and inflammatory factors in ox-LDL-treated HUVECs were reversed by the treatments with integrinβ1-siRNA or the JNK inhibitor. We also found that integrinβ1-siRNA decreased the protein expression of GTP-RhoA and p-JNK, while RhoA inhibitor partially reduced the upregulated expression of p-JNK induced by Gal-3. In conclusion, our finding suggests that Gal-3 exacerbates ox-LDL-mediated endothelial injury by inducing inflammation via integrin β1-RhoA-JNK signaling activation.     

Bacteriostatic stimulus of meropenem on allelochemical-metabolizing Burkholderia sp. LS-044 mitigates ferulic acid autotoxicity in rice (Oryza sativa ssp. japonica cv. Tainung 71)

Plant and Soil - To screen plant-associated Burkholderia strains for plant probiotic traits including allelochemical metabolism and understand their role on rice allelopathy using a...     

Reversal of docosahexaenoic acid deficiency in the rat brain, retina, liver, and serum

The loss of docosahexaenoic acid (DHA) from the retina or brain has been associated with a loss in nervous-system function in experimental animals, as well as in human infants fed vegetable oil-based formulas. The reversibility of the loss of DHA and the compensation by an increase in the n-6 docosapentaenoic acid (DPAn-6) was studied in young adult rats. Long-Evans rats were subjected to a very low level of n-3 fatty acids through two generations. The F2 generation, n-3-deficient animals at 7 weeks of age were provided a repletion diet containing both alpha-linolenate and DHA. A separate group of F2 generation rats had been maintained on an n-3-adequate diet of the same composition. Tissues from the brain, retina, liver, and serum were collected on weeks 0, 1, 2, 4, and 8 from both groups of animals. The concentrations of DHA, DPAn-6, and other fatty acids were determined and the rate of recovery and length of time needed to complete DHA recovery were determined for each tissue. The DHA level in the brain at 1 and 2 weeks after diet reversal was only partially recovered, rising to approximately 20% and 35%, respectively, of the n-3-adequate group level. Full recovery was not obtained until 8 weeks after initiation of the repletion diet. Although the initial rate of retinal DHA accretion was greater than that of brain DHA, the half-time for DHA recovery was only marginally greater. On the other hand, the levels of DHA in the serum and liver were approximately 90% and 100% replaced, respectively, within 2 weeks of diet reversal. A consideration of the total amounts and time courses of DHA repleted in the nervous system compared with the liver and circulation suggests that transport-related processes may limit the rate of DHA repletion in the retina and brain.-- Moriguchi, T., J. Loewke, M. Garrison, J. N. Catalan, N. Salem, Jr. Reversal of docosahexaenoic acid deficiency in the rat brain, retina, liver, and serum. J. Lipid Res. 2001. 42: 419--427.     

Correlation of observed fold frequency with the occurrence of local structural motifs

It is well known that some protein folds (superfolds) occur very frequently. We show that compared to other folds, most superfold structures have a higher proportion of their alpha-helical or beta-strand residues in one of three basic units of supersecondary structure (alpha-hairpin, beta-hairpin or betaalphabeta-unit). Furthermore, by taking into consideration two more complex motifs, the four-stranded Greek-key (beta4) and the betaalpha-Greek key (betaalphabetabeta), we demonstrate that the remaining superfold structures contain many of these higher order units of three-dimensional packing. The implications of these results for folding are discussed.     

Characterization and analysis of antibiotic activity of some aquatic actinomycetes

Nine different isolates of aquatic actinomycetes identified as Streptomyces spp. were studied for their morphological and cultural characteristics. One of these isolates (C4-S, Streptomyces violaceusniger) was extensively studied for its inhibitory effect against a wide range of Gram-positive, Gram-negative bacteria, Mycobacterium vaccae ATCC 29678, Candida albicans and several food associated filamentous fungi and yeasts. Most of these were characterized by flexous sporophore morphology and their inability to produce cultural pigments. Bioassay results indicated that S. violaceusniger of 10 days culture age was highly active against Gram-positive cocci and bacilli with an inhibition zone of 16-25 mm, and slightly active against M. vaccae ATCC 29678 with an inhibition zone of 5-10 mm. The inhibitory effect was slight against Escherichia coli, Aspergillus niger 1 and C. albicans with an inhibition zone of 8-10 mm for each of them. There was no inhibitory effect of S. violaceusniger against other Gram-negative bacteria, filamentous fungi and yeast. The nature of the active molecule produced by S. violaceusniger showed a maximum absorption in the UV region at 210-260 nm.