Apigenin shows synergistic anticancer activity with curcumin by binding at different sites of tubulin

Apigenin, a natural flavone, present in many plants sources, induced apoptosis and cell death in lung epithelium cancer (A549) cells with an IC₅₀ value of 93.7 ± 3.7 μM for 48 h treatment. Target identification investigations using A549 cells and also in cell-free system demonstrated that apigenin depolymerized microtubules and inhibited reassembly of cold depolymerized microtubules of A549 cells. Again apigenin inhibited polymerization of purified tubulin with an IC₅₀ value of 79.8 ± 2.4 μM. It bounds to tubulin in cell-free system and quenched the intrinsic fluorescence of tubulin in a concentration- and time-dependent manner. The interaction was temperature-dependent and kinetics of binding was biphasic in nature with binding rate constants of 11.5 × 10⁻⁷ M⁻¹ s⁻¹ and 4.0 × 10⁻⁹ M⁻¹ s⁻¹ for fast and slow phases at 37 °C, respectively. The stoichiometry of tubulin–apigenin binding was 1:1 and binding the binding constant (K_d) was 6.08 ± 0.096 μM. Interestingly, apigenin showed synergistic anti-cancer effect with another natural anti-tubulin agent curcumin. Apigenin and curcumin synergistically induced cell death and apoptosis and also blocked cell cycle progression at G₂/M phase of A549 cells. The synergistic activity of apigenin and curcumin was also apparent from their strong depolymerizing effects on interphase microtubules and inhibitory effect of reassembly of cold depolymerized microtubules when used in combinations, indicating that these ligands bind to tubulin at different sites. In silico modeling suggested apigenin bounds at the interphase of α–β-subunit of tubulin. The binding site is 19 Å in distance from the previously predicted curcumin binding site. Binding studies with purified protein also showed both apigenin and curcumin can simultaneously bind to purified tubulin. Understanding the mechanism of synergistic effect of apigenin and curcumin could be helped to develop anti-cancer combination drugs from cheap and readily available nutraceuticals.     

Harnessing mesenchymal stem cell secretome: Effect of extracellular matrices on proangiogenic signaling

The low engraftment and retention rate of mesenchymal stem cells (MSCs) at the target site indicates that the potential benefits of MSC-based therapies can be attributed to their paracrine signaling. In this study, the extracellular matrices (ECMs) deposited by bone marrow-derived human MSCs in the presence and absence of ascorbic acid was characterized. MSCs were seeded on top of decellularized ECM (dECM) and the concentrations of proangiogenic and antiangiogenic molecules released in culture (conditioned) media was compared. Effects of ECM derived from MSCs with different passage numbers on MSC secretome was also investigated. Our study revealed that the expression of proangiogenesis-related factors were upregulated when MSCs were harvested on dECMs, irrespective of media supplementation, as compared with those cultured on tissue culture plates. In addition, dECM generated in the presence of ascorbic acid promoted the expression of proangiogenic molecules as compared with dECM-derived in absence of media supplementation. Further, it was observed that the effectiveness of dECM to stimulate proangiogenic signaling of MSCs was reduced as cell passage number was increased from P3 to P5. The proliferation as well as capillary morphogenesis of human umbilical vein endothelial cells (HUVECs) in the presence of conditioned media were enhanced compared with the normal HUVECs culture media. These data indicate that the secretory signatures of MSCs and consequently, the therapeutic efficacy of MSCs can be regulated by presentation of dECM composition and variation of its composition.     

Archaeal flagellar ATPase motor shows ATP-dependent hexameric assembly and activity stimulation by specific lipid binding

Microbial motility frequently depends on flagella or type?IV pili. Using recently developed archaeal genetic tools, archaeal flagella and its assembly machinery have been identified. Archaeal flagella are functionally similar to bacterial flagella and their assembly systems are homologous with type?IV pili assembly systems of Gram-negative bacteria. Therefore elucidating their biochemistry may result in insights in both archaea and bacteria. FlaI, a critical cytoplasmic component of the archaeal flagella assembly system in Sulfolobus acidocaldarius, is a member of the type?II/IV secretion system ATPase superfamily, and is proposed to be bi-functional in driving flagella assembly and movement. In the present study we show that purified FlaI is a Mn2+-dependent ATPase that binds MANT-ATP [2'-/3'-O-(N'- methylanthraniloyl)adenosine-5'-O-triphosphate] with a high affinity and hydrolyses ATP in a co-operative manner. FlaI has an optimum pH and temperature of 6.5 and 75?°C for ATP hydrolysis. Remarkably, archaeal, but not bacterial, lipids stimulated the ATPase activity of FlaI 3-4-fold. Analytical gel filtration indicated that FlaI undergoes nucleotide-dependent oligomerization. Furthermore, SAXS (small-angle X-ray scattering) analysis revealed an ATP-dependent hexamerization of FlaI in solution. The results of the present study report the first detailed biochemical analyses of the motor protein of an archaeal flagellum.     

Inter-subunit recognition and manifestation of segmental mobility in Escherichia coli RNA polymerase: a case study with omega-beta' interaction

Omega (omega), consisting of 91 amino acids, is the smallest of all the Escherichia coli RNA polymerase subunits and is organized into an N-terminal domain of 53 amino acids followed by an unstructured tail in the C-terminal region. Our earlier experiments have shown a chaperone-like function of omega in which it helps to maintain beta' in a correct conformation and recruit it to the alpha(2)beta subassembly to form a functional core enzyme (alpha(2)betabeta'omega). The X-ray structure analysis of Thermus aquaticus core RNA polymerase suggests that two regions of omega latch onto the N-terminal and C-terminal ends of the beta'-subunit. In the present study we have monitored the conformational changes in beta' as the denatured protein is refolded in the presence and absence of omega using tryptophan fluorescence emission of beta' as well as acrylamide quenching of Trp fluorescence. Results indicate that the presence of stoichiometric amounts of omega is helpful in beta' refolding. We have also monitored the behavior of the C-terminal tail of omega by engineering three cysteine residues at three different sites in omega and subsequently labeling them with a sulphydryl-specific fluorescent probe. Fluorescence anisotropy measurements of the labeled protein indicate that the C-terminal domain of omega is mobile in the free protein and gets restrained in the presence of beta'. Calculations on side-chain interactions show that out of the three mutated positions, two have near neighbourhood interactions only with side-chains in the beta' subunit whereas the end of the C-terminal of omega, although it is restrained in the presence of beta', has no interacting partner within a 4-A radius.     

Insights into phytase-containing transgenic <Emphasis Type="Italic">Lemna minor</Emphasis> (L.) as a novel feed additive

This study assessed the effect of supplementation of novel transgenic phytase on growth performance and bone mineralization in Korean native broiler chickens. The experiment was designed using four dietary groups: those with a diet supplemented with (A) recombinant phytase, (B) transgenic phytase from the plant Lemna minor, (C) or wild-type L. minor as well as (D) a control group that was supplemented with commercially available feed. Three hundred 1-day-old Korean native broiler chicks were used and divided into these four dietary treatment groups having three replicates of 25 birds each (n?=?75). The results showed increases in growth performance and bone mineralization in Groups B and C; compared with Groups A and D. Hematological analyses revealed notable contrasts in erythrocyte sedimentation rate, red blood cell count, and hemoglobin levels among the experimental groups, whereas no impacts of dietary treatment were observed on total eosinophil, lymphocyte, heterophil, monocyte, and basophil levels. The relative expression profiling of candidate genes showed that the genes involved in growth response, meat quality, and P–Ca metabolism were significantly highly expressed in the phytase-supplemented groups. Hence, it is suggested that dietary supplementation with transgenic phytase plant L. minor for enhancing growth performance is a promising new approach in the broiler feed industry. To the best of our knowledge, we report here the most comprehensive analysis using a broiler model that provides a workable platform for further research on the cost-effective production of feed with different compositions that might be beneficial in the livestock feed industry.     

Improved nearest-neighbor parameters for the stability of RNA/DNA hybrids under a physiological condition

The stability of Watson–Crick paired RNA/DNA hybrids is important for designing optimal oligonucleotides for ASO (Antisense Oligonucleotide) and CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)–Cas9 techniques. Previous nearest-neighbour (NN) parameters for predicting hybrid stability in a 1 M NaCl solution, however, may not be applicable for predicting stability at salt concentrations closer to physiological condition (e.g. ∼100 mM Na⁺ or K⁺ in the presence or absence of Mg²⁺). Herein, we report measured thermodynamic parameters of 38 RNA/DNA hybrids at 100 mM NaCl and derive new NN parameters to predict duplex stability. Predicted ΔG°₃₇ and T_m values based on the established NN parameters agreed well with the measured values with 2.9% and 1.1°C deviations, respectively. The new results can also be used to make precise predictions for duplexes formed in 100 mM KCl or 100 mM NaCl in the presence of 1 mM Mg²⁺, which can mimic an intracellular and extracellular salt condition, respectively. Comparisons of the predicted thermodynamic parameters with published data using ASO and CRISPR–Cas9 may allow designing shorter oligonucleotides for these techniques that will diminish the probability of non-specific binding and also improve the efficiency of target gene regulation.     

Effects of deletions in the carboxy-terminal hydrophobic region of herpes simplex virus glycoprotein gB on intracellular transport and membrane anchoring.

The gB glycoprotein of herpes simplex virus type 1 is involved in viral entry and fusion and contains a predicted membrane-anchoring sequence of 69 hydrophobic amino acids, which can span the membrane three times, near the carboxy terminus. To define the membrane-anchoring sequence and the role of this hydrophobic stretch, we have constructed deletion mutants of gB-1, lacking one, two, or three predicted membrane-spanning segments within the 69 amino acids. Expression of the wild-type and mutant glycoproteins in COS-1 cells show that mutant glycoproteins lacking segment 3 (amino acids 774 to 795 of the gB-1 protein) were secreted from the cells. Protease digestion and alkaline extraction of microsomes containing labeled mutant proteins further showed that segment 3 was sufficient for stable membrane anchoring of the glycoproteins, indicating that this segment may specify the transmembrane domain of the gB glycoprotein. Also, the mutant glycoproteins containing segment 3 were localized in the nuclear envelop, which is the site of virus budding. Deletion of any of the hydrophobic segments, however, affected the intracellular transport and processing of the mutant glycoproteins. The mutant glycoproteins, although localized in the nuclear envelope, failed to complement the gB-null virus (K082). These results suggest that the carboxy-terminal hydrophobic region contains essential structural determinants of the functional gB glycoprotein.     

Antimicrobial activity of Sesbania grandiflora flower polyphenol extracts on some pathogenic bacteria and growth stimulatory effect on the probiotic organism Lactobacillus acidophilus

Polyphenolic extracts (PE) of edible flower of Sesbania grandiflora were tested to evaluate its antimicrobial effect against some common pathogenic bacteria and growth promoting property against probiotic organism Lactobacillus acidophilus. The antimicrobial activity of S. grandiflora flower PE against selected pathogens was evaluated using both in vitro and in situ methods. In vitro studies suggested that PE has inhibitory effect against Staphylococcus aureus, Shigella flexneri 2a, Salmonella Typhi, Escherichia coli and Vibrio cholerae. The gram-positive organism S. aureus was the most sensitive organism to PE and minimum inhibitory concentration (MIC) was found to be 0.013 mg/mL where as the MIC of PE against V. cholerae was the highest (0.25 mg/mL). On the other hand PE showed growth promoting effect on the common probiotic bacterium L. acidophilus. The major finding was that S. grandiflora PE induced a significant biomass increase of L. acidophilus grown in liquid culture media. PE showed reduction of S. aureus growth in food (fish) during storage at 10°C. High performance liquid chromatography analysis showed that rutin, a major flavonoid of the PE diminished in the culture medium MRS broth with the growth of L. acidophilus.     

Proteolysis of insulin-like growth factor binding proteins (IGFBPs) by calpain

Calpains are non-lysosomal, Ca 2+ -dependent cysteine proteases, which are ubiquitously distributed across cell types and vertebrate species. The rules that govern calpain specificity have not yet been determined. To elucidate the cleavage pattern of calpains, we carried out calpain-induced proteolytic studies on the insulin-like growth factor binding proteins IGFBP-4 and -5. Proteolysis of IGFBPs is well characterized in numerous reports. Our results show that calpain cleavage sites are in the non-conserved unstructured regions of the IGFBPs. Compilation of the calpain-induced proteolytic cleavage sites in several proteins reported in the literature, together with our present study, has not revealed clear preferences for amino acid sequences. We therefore conclude that calpains seem not to recognize amino acid sequences, but instead cleave with low sequence specificity at unstructured or solvent-exposed fragments that connect folded, stable domains of target proteins.