Pharmacognostic Standardization, Physico- and Phytochemical Evaluation of Amaranthus Spinosus Linn. Root

Amaranthus spinosus Linn. (Amaranthaceae) is found throughout India. This tree species has been of interest to researchers because it is a medicinal plant employed in the Indian traditional system of medicine. Pharmacognostic standardization; physico-and phytochemical evaluation of the roots of Amaranthus spinosus was carried out, to determine its macro-and microscopical characters, and also some of its quantitative standards. Microscopical studies were done by using the trinocular microscope. Total ash, water-soluble ash, acid-insoluble ash, sulfated ash values, and alcohol-and water-soluble extractive values were determined for physico-chemical evaluations. A preliminary phytochemical screening was also done to detect different phytoconstituents. Microscopically, the root showed cork, cortex, stellar region, and calcium oxalate crystals. Powder microscopy showed anamalous secondary growth in between the xylem vessels and Calcium Oxalate crystals in the cortex region. Total ash was approximately three times more than acid insoluble and water soluble ash. The ethanol soluble extractive was approximately the same as the water soluble extractive. Thin Layer Chromatography (TLC) of the Petroleum-ether extract using Benzene : Ethyl acetate (6 : 1), showed six spots. In the chloroform extract, using Benzene : Ethyl acetate (4 : 1) nine spots were seen, and in the ethanol extract, using Chloroform: Methanol (93 : 7), only four spots were observed, using Iodine vapor as a viewing medium. Phytochemically, the root exhibited terpenes, alkaloids, glycosides, and sugars. These findings might be useful to supplement information with regard to its identification parameters, which are assumed significant in the way of acceptability of herbal drugs, in the present scenario, which lacks regulatory laws to control the quality of herbal drugs

Pleiotropic effects of the vacuolar ABC transporter MLT1 of Candida albicans on cell function and virulence.

Among the several mechanisms that contribute to MDR (multidrug resistance), the overexpression of drug-efflux pumps belonging to the ABC (ATP-binding cassette) superfamily is the most frequent cause of resistance to antifungal agents. The multidrug transporter proteins Cdr1p and Cdr2p of the ABCG subfamily are major players in the development of MDR in Candida albicans Because several genes coding for ABC proteins exist in the genome of C. albicans, but only Cdr1p and Cdr2p have established roles in MDR, it is implicit that the other members of the ABC family also have alternative physiological roles. The present study focuses on an ABC transporter of C. albicans, Mlt1p, which is localized in the vacuolar membrane and specifically transports PC (phosphatidylcholine) into the vacuolar lumen. Transcriptional profiling of the mlt1∆/∆ mutant revealed a down-regulation of the genes involved in endocytosis, oxidoreductase activity, virulence and hyphal development. High-throughput MS-based lipidome analysis revealed that the Mlt1p levels affect lipid homoeostasis and thus lead to a plethora of physiological perturbations. These include a delay in endocytosis, inefficient sequestering of reactive oxygen species (ROS), defects in hyphal development and attenuated virulence. The present study is an emerging example where new and unconventional roles of an ABC transporter are being identified

The structural basis for regulation of the glutathione transporter Ycf1 by regulatory domain phosphorylation

Yeast Cadmium Factor 1 (Ycf1) sequesters heavy metals and glutathione into the vacuole to counter cell stress. Ycf1 belongs to the ATP binding cassette C-subfamily (ABCC) of transporters, many of which are regulated by phosphorylation on intrinsically-disordered domains. The regulatory mechanism of phosphorylation is still poorly understood. Here, we report two cryo-EM structures of Ycf1 at 3.4 Å and 4.0 Å resolution in inward-facing open conformations that capture previously unobserved ordered states of the intrinsically disordered regulatory domain (R-domain). R-domain phosphorylation is clearly evident and induces a topology promoting electrostatic and hydrophobic interactions with Nucleotide Binding Domain 1 (NBD1) and the Lasso motif. These interactions stay constant between the structures and are related by rigid body movements of the NBD1/R-domain complex. Biochemical data further show R-domain phosphorylation reorganizes the Ycf1 architecture and is required for maximal ATPase activity. Together, we provide insights into how R-domains control ABCC transporter activity. © 2022, The Author(s).Open access journalThis item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Inositol Phosphoryl Transferase, Ipt1, Is a Critical Determinant of Azole Resistance and Virulence Phenotypes in Candida glabrata

In this study, we have specifically blocked a key step of sphingolipid (SL) biosynthesis in Candida glabrata by disruption of the orthologs of ScIpt1 and ScSkn1. Based on their close homology with S. cerevisiae counterparts, the proteins are predicted to catalyze the addition of a phosphorylinositol group onto mannosyl inositolphosphoryl ceramide (MIPC) to form mannosyl diinositolphosphoryl ceramide (M(IP)2C), which accounts for the majority of complex SL structures in S. cerevisiae membranes. High throughput lipidome analysis confirmed the accumulation of MIPC structures in ΔCgipt1 and ΔCgskn1 cells, albeit to lesser extent in the latter. Noticeably, ΔCgipt1 cells showed an increased susceptibility to azoles; however, ΔCgskn1 cells showed no significant changes in the drug susceptibility profiles. Interestingly, the azole susceptible phenotype of ΔCgipt1 cells seems to be independent of the ergosterol content. ΔCgipt1 cells displayed altered lipid homeostasis, increased membrane fluidity as well as high diffusion of radiolabeled fluconazole (3H-FLC), which could together influence the azole susceptibility of C. glabrata. Furthermore, in vivo experiments also confirmed compromised virulence of the ΔCgipt1 strain. Contrarily, specific functions of CgSkn1 remain unclear. © 2022 by the authors. Licensee MDPI, Basel, Switzerland.Open access journalThis item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Azole resistance in a <i>Candida albicans</i> mutant lacking the ABC transporter <i>CDR6/ROA1</i> depends on TOR signaling.

ATP-binding cassette (ABC) transporters help export various substrates across the cell membrane and significantly contribute to drug resistance. However, a recent study reported an unusual case in which the loss of an ABC transporter in &lt;i&gt;Candida albicans&lt;/i&gt; , orf19.4531 (previously named ROA1), increases resistance against antifungal azoles, which was attributed to an altered membrane potential in the mutant strain. To obtain further mechanistic insights into this phenomenon, here we confirmed that the plasma membrane-localized transporter (renamed &lt;i&gt;CDR6/ROA1&lt;/i&gt; for consistency with &lt;i&gt;C. albicans&lt;/i&gt; nomenclature) could efflux xenobiotics such as berberine, rhodamine 123, and paraquat. Moreover, a &lt;i&gt;CDR6/ROA1&lt;/i&gt; null mutant, NKKY101, displayed increased susceptibility to these xenobiotics. Interestingly, fluorescence recovery after photobleaching (FRAP) results indicated that NKKY101 mutant cells exhibited increased plasma membrane rigidity, resulting in reduced azole accumulation and contributing to azole resistance. Transcriptional profiling revealed that ribosome biogenesis genes were significantly up-regulated in the NKKY101 mutant. As ribosome biogenesis is a well-known downstream phenomenon of target of rapamycin (TOR1) signaling, we suspected a link between ribosome biogenesis and TOR1 signaling in NKKY101. Therefore, we grew NKKY101 cells on rapamycin and observed TOR1 hyperactivation, which leads to Hsp90-dependent calcineurin stabilization and thereby increased azole resistance. This &lt;i&gt;in vitro&lt;/i&gt; finding was supported by &lt;i&gt;in vivo&lt;/i&gt; data from a mouse model of systemic infection in which NKKY101 cells led to higher fungal load after fluconazole challenge than wild-type cells. Taken together, our study uncovers a mechanism of azole resistance in &lt;i&gt;C. albicans&lt;/i&gt; , involving increased membrane rigidity and TOR signaling