Depigmenting effect of Xanthohumol from hop extract in MNT-1 human melanoma cells and normal human melanocytes

Xanthohumol (XH) is the most abundant prenylated flavonoid found in the hop plant (Humulus lupulus L.) and has previously been shown to have depigmenting effects in B16F10 mouse melanoma cells; however, studies of its depigmenting efficacy in human melanocytes are still lacking. In this work, we explored the effects of XH on melanogenesis in MNT-1 human melanoma cells and normal human melanocytes from darkly-pigmented skin (HEM-DP). XH was screened for cytotoxicity over 48 h, and subsequently tested on melanogenesis in MNT-1 cells. XH was further tested in HEM-DP cells for melanin synthesis and melanosome export; dendricity was quantitated to assess effects on melanosome export. Melanosome degradation was studied in human keratinocytes (HaCaT). Our results showed that XH inhibited melanin synthesis in MNT-1 cells at 30 μM but increased intracellular tyrosinase activity without affecting ROS levels. In HEM-DP cells, XH robustly suppressed cellular tyrosinase activity at nontoxic concentrations (2.5–5 μM) without any effect on melanin synthesis. However, XH inhibited melanosome export by reducing dendrite number and total dendrite length. Further testing in HaCaT cells demonstrated that XH induced melanosome degradation at low micromolar concentrations without any cytotoxicity. In summary, our results demonstrate that XH at low micromolar concentrations might hold promise as a potent inhibitor of human pigmentation by primarily targeting melanin export and melanin degradation. Further studies to elucidate the signaling mechanisms of action of melanosome export inhibition by XH and in vivo efficacy are warranted.     

Corticotropin-releasing Factor Receptor 2 Mediates Sex-Specific Cellular Stress Responses

Although females suffer twice as much as males from stress-related disorders, sex-specific participating and pathogenic cellular stress mechanisms remain uncharacterized. Using corticotropin-releasing factor receptor 2–deficient (Crhr2^−/− ) and wild-type (WT) mice, we show that CRF receptor type 2 (CRF₂) and its high-affinity ligand, urocortin 1 (Ucn1), are key mediators of the endoplasmic reticulum (ER) stress response in a murine model of acute pancreatic inflammation. Ucn1 was expressed de novo in acinar cells of male, but not female WT mice during acute inflammation. Upon insult, acinar Ucn1 induction was markedly attenuated in male but not female Crhr2^−/− mice. Crhr₂^−/− mice of both sexes show exacerbated acinar cell inflammation and necrosis. Electron microscopy showed mild ER damage in WT male mice and markedly distorted ER structure in Crhr2^−/− male mice during pancreatitis. WT and Crhr2^−/− female mice showed similarly distorted ER ultrastructure that was less severe than distortion seen in Crhr2^−/− male mice. Damage in ER structure was accompanied by increased ubiquitination, peIF2, and mistargeted localization of vimentin in WT mice that was further exacerbated in Crhr2^−/− mice of both sexes during pancreatitis. Exogenous Ucn1 rescued many aspects of histological damage and cellular stress response, including restoration of ER structure in male WT and Crhr2^−/−mice, but not in females. Instead, females often showed increased damage. Thus, specific cellular pathways involved in coping and resolution seem to be distinct to each sex. Our results demonstrate the importance of identifying sex-specific pathogenic mechanisms and their value in designing effective therapeutics.     

Targeting RET to induce medullary thyroid cancer cell apoptosis: an antagonistic interplay between PI3K/Akt and p38MAPK/caspase-8 pathways

Mutations in REarranged during Transfection (RET) receptor tyrosine, followed by the oncogenic activation of RET kinase is responsible for the development of medullary thyroid carcinoma (MTC) that responds poorly to conventional chemotherapy. Targeting RET, therefore, might be useful in tailoring surveillance of MTC patients. Here we showed that theaflavins, the bioactive components of black tea, successfully induced apoptosis in human MTC cell line, TT, by inversely modulating two molecular pathways: (i) stalling PI3K/Akt/Bad pathway that resulted in mitochondrial transmembrane potential (MTP) loss, cytochrome-c release and activation of the executioner caspases-9 and -3, and (ii) upholding p38MAPK/caspase-8/caspase-3 pathway via inhibition of Ras/Raf/ERK. Over-expression of either constitutively active myristoylated-Akt-cDNA (Myr-Akt-cDNA) or dominant-negative-caspase-8-cDNA (Dn-caspase-8-cDNA) partially blocked theaflavin-induced apoptosis, while co-transfection of Myr-Akt-cDNA and Dn-caspase-8-cDNA completely eradicated the effect of theaflavins thereby negating the possibility of existence of other pathways. A search for the upstream signaling revealed that theaflavin-induced disruption of lipid raft caused interference in anchorage of RET in lipid raft that in turn stalled phosphorylation of Ras and PI3Kinase. In such anti-survival cellular micro-environment, pro-apoptotic signals were triggered to culminate into programmed death of MTC cell. These findings not only unveil a hitherto unexplained mechanism underlying theaflavin-induced MTC death, but also validate RET as a promising and potential target for MTC therapy.     

185 Vascular endothelial growth factor receptor-2 (VEGFR2): structure and functional relationship

Vascular endothelial growth factor (VEGF), is expressed in the vicinity of sprouting vessels and its receptor (VEGF-R2/Flk-1/kdr) on the angioblasts and new vessels, and both are required for vasculogenesis and angiogenesis. VEGFR2, also called as KDR or Flk-1, is identified as an early marker for endothelial cell progenitors, whose expression is restricted to endothelial cells in vivo. VEGFR2 consists of extracellular (7-Ig-like sub-domains), transmembrane and cytoplasmic domains. In order to understand the structure–functional relationship and signal transduction process of VEGFR2, we have examined their amino acid sequences from a wide range of species including mammals, birds, Zebrafish and also computed the phylogenetic tree, secondary and domain structures. Phylogeny constructed using Maximum Parsimony tree software MEGA-5 version suggested an interesting sequence similarity between Zebrafish and Gallus, closeness between human, rat, horse and pig. Strong homology in amino acids sequences was observed between the species, such as human, Macaca mulatta, gorilla, etc, and small variations in Zebrafish and zebrafinch. The Arg and Asp residues which are involved in forming salt bridges are evolutionarily conserved from Zebrafish to human in D7 domain of VEGFR2, indicating their functional importance in VEGFR activity. Amino acids, tyrosine in the extracellular loops and cysteines involved in disulphide bridges of VEGFR2, are highly conserved suggesting their importance during ligand binding, the details of which will be discussed.     

Combating biotic stresses in plants by synthetic microbial communities: Principles,applications and challenges

Presently, agriculture worldwide is facing the major challenge of feeding the increasing population sustainably. The conventional practices have not only failed to meet the projected needs, but also led to tremendous environmental consequences. Hence, to ensure a food-secure and environmentally sound future, the major thrust is on sustainable alternatives. Due to challenges associated with conventional means of application of biocontrol agents in the management of biotic stresses in agroecosystems, significant transformations in this context are needed. The crucial role played by soil microbiome in efficiently and sustainably managing the agricultural production has unfolded a newer approach of rhizosphere engineering that shows immense promise in mitigating biotic stresses in an eco-friendly manner. The strategy of generating synthetic microbial communities (SynComs), by integrating omics approaches with traditional techniques of enumeration and in-depth analysis of plant–microbe interactions, is encouraging. The review discusses the significance of the rhizospheric microbiome in plant's fitness, and its manipulation for enhancing plant attributes. The focus of the review is to critically analyse the potential tools for the design and utilization of SynComs as a sustainable approach for rhizosphere engineering to ameliorate biotic stresses in plants. Furthermore, based on the synthesis of reports in the area, we have put forth possible solutions to some of the critical issues that impair the large-scale application of SynComs in agriculture.     

Expression of recombinant zeta-crystallin in Escherichia coli with the help of GroEL/ES and its purification

zeta-Crystallin is a taxon-specific crystallin found in the eye lens of guinea pig and other hystricomorph rodents and camelids. It is an NADPH:quinone oxidoreductase and is also present in low amounts in other tissues where it might act as a detoxifying enzyme. A lens-specific promoter confers lens-specific expression of the gene in high amounts where it is speculated to play a structural role in maintaining the transparency of the lens ensemble. A deletion mutation leads to autosomal dominant congenital cataract and also results in the loss of NADPH binding. In order to perform structural studies with the protein with an aim to delineate the cause of cataract in these mutant guinea pigs, recombinant zeta-crystallin was cloned and expressed in Escherichia coli. The overexpression of the protein in E. coli resulted in a major fraction of it partitioning into inclusion bodies. The co-overexpression of the bacterial chaperone system GroEL/ES along with zeta-crystallin could significantly enhance the yield of soluble protein. Active zeta-crystallin could then be purified from the E. coli using Mono Q anion exchange FPLC and was found to be identical to the native zeta-crystallin isolated from the guinea pig lens with respect to size, spectral properties, and activity.