A New Hydroxylated Nonaprenylhydroquinone from the Mediterranean Marine Sponge Sarcotragus spinosulus

Chemical investigation of the Mediterranean sponge Sarcotragus spinosulus led to the isolation of a new hydroxylated nonaprenylhydroquinone, along with two known metabolites, hepta- and octaprenylhydroquinones. The structure of the new metabolite was assigned by extensive 1D and 2D NMR analyses and MS studies. The antileukemic effect of the three compounds towards the chronic myelogenous leukemia (CML) cells line K562 was also evaluated

Combined metabolome and transcriptome profiling provides new insights into diterpene biosynthesis in S. pomifera glandular trichomes

Background: Salvia diterpenes have been found to have health promoting properties. Among them, carnosic acid and carnosol, tanshinones and sclareol are well known for their cardiovascular, antitumor, antiinflammatory and antioxidant activities. However, many of these compounds are not available at a constant supply and developing biotechnological methods for their production could provide a sustainable alternative. The transcriptome of S. pomifera glandular trichomes was analysed aiming to identify genes that could be used in the engineering of synthetic microbial systems. Results: In the present study, a thorough metabolite analysis of S. pomifera leaves led to the isolation and structure elucidation of carnosic acid-family metabolites including one new natural product. These labdane diterpenes seem to be synthesized through miltiradiene and ferruginol. Transcriptomic analysis of the glandular trichomes from the S. pomifera leaves revealed two genes likely involved in miltiradiene synthesis. Their products were identified and the corresponding enzymes were characterized as copalyl diphosphate synthase (SpCDS) and miltiradiene synthase (SpMilS). In addition, several CYP-encoding transcripts were identified providing a valuable resource for the identification of the biosynthetic mechanism responsible for the production of carnosic acid-family metabolites in S. pomifera. Conclusions: Our work has uncovered the key enzymes involved in miltiradiene biosynthesis in S. pomifera leaf glandular trichomes. The transcriptomic dataset obtained provides a valuable tool for the identification of the CYPs involved in the synthesis of carnosic acid-family metabolites.General Secretariat of Research and Technology (GSRT) {[}09-SYN-23-879]; grant SEE-ERA. NET PLUS {[}ERA 64/01]; grant KRIPIS {[}MIS 448840

Characterizing the Anti-HIV Activity of Papuamide A

Papuamide A is representative of a class of marine derived cyclic depsipeptides, reported to have cytoprotective activity against HIV-1 in vitro. We show here that papuamide A acts as an entry inhibitor, preventing human immunodeficiency virus infection of host cells and that this inhibition is not specific to R5 or X4 tropic virus. This inhibition of viral entry was determined to not be due to papuamide A binding to CD4 or HIV gp120, the two proteins involved in the cell-virus recognition and binding. Furthermore, papuamide A was able to inhibit HIV pseudotype viruses expressing envelope glycoproteins from vesicular stomatitis virus or amphotropic murine leukemia virus indicating the mechanism of viral entry inhibition is not HIV-1 envelope glycoprotein specific. Time delayed addition studies with the pseudotyped viruses show that papuamide A inhibits viral infection only at the initial stage of the viral life cycle. Additionally, pretreatment studies revealed that the virus, and not the cell, is the target of papuamide A’s action. Together, these results suggest a direct virucidal mechanism of HIV-1 inhibition by papuamide A. We also demonstrate here that the other papuamides (B-D) are able to inhibit viral entry indicating that the free amino moiety of 2,3-diaminobutanoic acid residue is not required for the virucidal activity

Inhibition of HIV-1 entry by extracts derived from traditional Chinese medicinal herbal plants

<p>Abstract</p> <p>Background</p> <p>Highly active anti-retroviral therapy (HAART) is the current HIV/AIDS treatment modality. Despite the fact that HAART is very effective in suppressing HIV-1 replication and reducing the mortality of HIV/AIDS patients, it has become increasingly clear that HAART does not offer an ultimate cure to HIV/AIDS. The high cost of the HAART regimen has impeded its delivery to over 90% of the HIV/AIDS population in the world. This reality has urgently called for the need to develop inexpensive alternative anti-HIV/AIDS therapy. This need has further manifested by recent clinical trial failures in anti-HIV-1 vaccines and microbicides. In the current study, we characterized a panel of extracts of traditional Chinese medicinal herbal plants for their activities against HIV-1 replication.</p> <p>Methods</p> <p>Crude and fractionated extracts were prepared from various parts of nine traditional Chinese medicinal herbal plants in Hainan Island, China. These extracts were first screened for their anti-HIV activity and cytotoxicity in human CD4+ Jurkat cells. Then, a single-round pseudotyped HIV-luciferase reporter virus system (HIV-Luc) was used to identify potential anti-HIV mechanisms of these extracts.</p> <p>Results</p> <p>Two extracts, one from <it>Euphorbiaceae</it>, <it>Trigonostema xyphophylloides </it>(TXE) and one from <it>Dipterocarpaceae</it>, <it>Vatica astrotricha </it>(VAD) inhibited HIV-1 replication and syncytia formation in CD4+ Jurkat cells, and had little adverse effects on host cell proliferation and survival. TXE and VAD did not show any direct inhibitory effects on the HIV-1 RT enzymatic activity. Treatment of these two extracts during the infection significantly blocked infection of the reporter virus. However, pre-treatment of the reporter virus with the extracts and treatment of the extracts post-infection had little effects on the infectivity or gene expression of the reporter virus.</p> <p>Conclusion</p> <p>These results demonstrate that TXE and VAD inhibit HIV-1 replication likely by blocking HIV-1 interaction with target cells, i.e., the interaction between gp120 and CD4/CCR5 or gp120 and CD4/CXCR4 and point to the potential of developing these two extracts to be HIV-1 entry inhibitors.</p

Metabolites with Antioxidant Activity from Marine Macroalgae

Reactive oxygen species (ROS) attack biological molecules, such as lipids, proteins, enzymes, DNA, and RNA, causing cellular and tissue damage. Hence, the disturbance of cellular antioxidant homeostasis can lead to oxidative stress and the onset of a plethora of diseases. Macroalgae, growing in stressful conditions under intense exposure to UV radiation, have developed protective mechanisms and have been recognized as an important source of secondary metabolites and macromolecules with antioxidant activity. In parallel, the fact that many algae can be cultivated in coastal areas ensures the provision of sufficient quantities of fine chemicals and biopolymers for commercial utilization, rendering them a viable source of antioxidants. This review focuses on the progress made concerning the discovery of antioxidant compounds derived from marine macroalgae, covering the literature up to December 2020. The present report presents the antioxidant potential and biogenetic origin of 301 macroalgal metabolites, categorized according to their chemical classes, highlighting the mechanisms of antioxidative action when known

The plastidic protease and its role in LHCII degradation

In the current PhD thesis the nature of plastidic LHCII (Light Harvesting Complex serving PSII) protease and its role in biogenesis and photosynthetic membrane function was studied. The study was conducted by using mainly biochemical and also molecular biology techniques (isolation, separation and characterization of macromolecules (proteins/DNA)).It was found that intact etioplasts of bean (Phaseolus vulgaris) plants exhibit proteolytic activity against the exogenously added apoprotein of the light-harvesting pigment-protein complex serving photosystem II that increases as etiolation is prolonged. The activity increases in the membrane fraction but not in the stroma, where it remains low and constant and is mainly directed against LHCII and protochlorophyllide oxidoreductase. The thylakoid proteolytic activity, which is low in etioplasts of 6-d-old etiolated plants, increases in plants pretreated with a pulse of light or exposed to intermittent-light (ImL) cycles, but decreases during prolonged exposure to continuous light, coincident with chlorophyll (Chl) accumulation. To distinguish between the control of Chl and/or development on proteolytic activity, we used plants exposed to ImL cycles of varying dark-phase durations. In ImL plants exposed to an equal number of ImL cycles with short or long dark intervals (i.e. equal Chl accumulation but different developmental stage) proteolytic activity increased with the duration of the dark phase. In plants exposed to ImL for equal durations to such light-dark cycles (i.e. different Chl accumulation but same developmental stage) the proteolytic activity was similar. These results suggest that the protease, which is free to act under limited Chl accumulation, is dependent on the developmental stage of the chloroplast, and give a clue as to why plants in ImL with short dark intervals contain LHCII, whereas those with long dark intervals possess only photosystem-unit cores and lack LHCII.The effect of Cd on chlorophyll (Chl) as well as on LHCII accumulation has also been examined during the early stages of development in etiolated Phaseolus vulgaris leaves exposed to intermittent light-dark cycles. We found that at the Cd concentrations studied, both Chl and LHCII accumulation were drastically reduced, although the lithium dodecyl sulphate-solubilized total leaf protein level remained unaffected. However, on the basis of total chlorophyll present, the amount of stabilized LHCII was similar in both Cd-treated and nontreated samples. Additionally, the thylakoid-bound protease known to degrade LHCII was found to be inhibited by Cd treatment both in vivo and in vitro. Finally, Northern hybridization analysis indicated that Cd affects LHCII accumulation by reducing drastically the steady-state level of Lhcb transcripts.Στην παρούσα διδακτορική διατριβή μελετήθηκε η φύση της πρωτεάσης της LHCII (Light Harvesting Complex) αποπρωτεΐνης των χλωροπλαστών από φύλλα φασολιού (Phaseolus vulgaris) και ο ρυθμιστικός της ρόλος τόσο στην βιογένεση όσο και στην λειτουργία της φωτοσυνθετικής μεμβράνης. Η μελέτη αυτή πραγματοποιήθηκε με χρήση κυρίως βιοχημικών, αλλά και τεχνικών μοριακής βιολογίας (απομόνωση, διαχωρισμός και χαρακτηρισμός μακρομορίων (πρωτεϊνών/DNA)).Βρέθηκε ότι άθικτοι ωχροπλάστες φασολιού παρουσιάζουν πρωτεολυτική ενεργότητα ως προς εξωγενώς προστιθέμενη LHCII αποπρωτείνη, που αυξάνει παράλληλα με την ανάπτυξη του φυτού. Η ενεργότητα αυξάνει στο μεμβρανικό κλάσμα και όχι στο στρώμα, όπου εκεί παραμένει χαμηλή και σταθερή. Επομένως, η αύξηση που παρατηρούμε δεν οφείλεται σε μεταβαλλόμενη πρόσδεση της πρωτεάσης στα προθυλακοειδή κατά την ανάπτυξη, αλλά πιθανότατα αντικατοπτρίζει την de novo σύνθεση του πρωτεολυτικού συστήματος. H πρωτεολυτική ενεργότητα των θυλακοειδών είναι χαμηλή σε ωχροπλάστες ηλικίας 6-ημερών και αυξάνει σε φυτά που εκθέτονται σε μια αναλαμπή φωτός ή σε κύκλους περιοδικού φωτισμού, αλλά μειώνεται μετά από παρατεταμένη έκθεση σε συνεχή φωτισμό, παράλληλα με την συσσώρευση της χλωροφύλλης. Το φαινόμενο όμως αυτό εξαλείφεται όταν τα θυλακοειδή διαλυτοποιηθούν σε ΤΧ-100. Αντίθετα, η διαλυτοποίηση σε TX-100 των προθυλακοειδών και των πρωτογενών θυλακοειδών δεν επιδρά στην πρωτεολυτική τους ενεργότητα ως προς εξωγενή LHCII αποπρωτεΐνη. Επιπλέον, η προσθήκη ΤΧ-100 διαλυτοποιημένων ώριμων θυλακοειδών (πλούσιων σε χλωροφύλλη) σε αδιάλυτα πρωτογενή θυλακοειδή δεν έχει καμία επίδραση στην πρωτεολυτική ενεργότητα των δεύτερων. Συμπεραίνουμε επομένως, ότι η μείωση της ενεργότητας στα θυλακοειδή φυτών που εκθέτονται στον συνεχή φωτισμό, οφείλεται σε παρεμπόδιση της πρωτεολυτικής δράσης λόγω οργάνωσης του υποστρώματος και όχι λόγω αναστολής της πρωτεολυτικής δράσης από την χλωροφύλλη. Επομένως, ο σχηματισμός χλωροφυλλοπρωτεϊνικού συμπλόκου προστατεύει την LHCII αποπρωτεΐνη από την πρωτεάση.Βρέθηκε επίσης ότι φυτά που εκθέτονται σε περιοδικό φωτισμό και δέχονται τον ίδιο αριθμό αναλαμπών, έχοντας όμως διαφορετική διάρκεια σκοτόφασης (ίδια συσσώρευση χλωροφύλλης, διαφορετικό στάδιο ανάπτυξης), παρουσιάζουν μεγαλύτερη ενεργότητα όσο μεγαλώνει η διάρκεια της σκοτόφασης (περισσότερο αναπτυγμένα). Φυτά που βρίσκονται στην ίδια αναπτυξιακή κατάσταση αλλά έχουν συσσωρεύσει διαφορετικά ποσά χλωροφύλλης έχουν την ίδια περίπου πρωτεολυτική ενεργότητα. Τα παραπάνω αποτελέσματα υποδεικνύουν ότι η πρωτεάση, η οποία μπορεί και δρα σε συνθήκες περιορισμένης συσσώρευσης χλωροφύλλης και εξαρτάται από το στάδιο ανάπτυξης του φυτού, παίζει καθοριστικό ρόλο στην σταθεροποίηση της LHCII αποπρωτεΐνης. Επίσης μελετήθηκε η επίδραση του Cd2+ στην συσσώρευση χλωροφύλλης και στην σταθεροποίηση της LHCII αποπρωτεΐνης κατά την ανάπτυξη των φυτών σε περιοδικό φωτισμό. Βρέθηκε ότι χορήγηση in vivo διαλυμάτων Cd2+ σε φυτά που αναπτύσσονται σε συνθήκες περιοδικού φωτισμού προκαλεί μείωση της ποσότητας LHCII που σταθεροποιείται στα πρωτογενή θυλακοειδή. Στις συνθήκες αυτές η συσσώρευση Chl μειώνεται, η ικανότητα πρωτεϊνικής σύνθεσης δεν επηρεάζεται, ενώ ο λόγος σταθεροποιημένης LHCII/Chl παραμένει σταθερός. Επιπλέον, η πρωτεολυτική ενεργότητα που αποδομεί την LHCII αποπρωτεΐνης των πρωτογενών θυλακοειδών αναστέλλεται παρουσία Cd2+ τόσο in vivo όσο και in vitro. Τέλος, βρέθηκε ότι η επίδραση της χορήγησης Cd2+ εκδηλώνεται στο μεταγραφικό στάδιο, προκαλώντας την μείωση του αριθμού των μεταγραφημάτων της LHCII

Natural products with anti-HIV activity from marine organisms.

In order to combat the human immunodeficiency virus (HIV), diverse strategies have been developed to research on compounds which can be developed as therapeutic agents. Screening of natural products derived from numerous species has afforded metabolites with significant antiviral activity against the HIV. The marine environment representing approximately half of the global biodiversity offers an enormous resource for novel compounds. Currently more than 150 natural products with promising levels of anti-HIV activity have been isolated following bioassay guided protocols from aqueous or organic extracts of marine organisms. Some of the most characteristic marinemetabolites that have exhibited significant anti-HIV activity on different biochemical assays designed for chemotherapeutic strategies are: Cyanovirin-N, a protein from a blue green alga; various sulfated polysaccharides extracted from seaweeds (i.e. Nothogenia fastigiata, Aghardhiella tenera); the peptides tachyplesin and polyphemusin, which are highly abundant in hemocyte debris of the horseshoe crabs Tachypleus tridentatus and Limulus polyphemus; sponge metabolites such as avarol, avarone, ilimaquinone and several phloroglucinols; and a number of metabolites from marine fungi such as equisetin, phomasetin and integric acid. Considering that number of unique metabolites that have been isolated from a small extent of the ocean&apos;s biological and chemical diversity, the oceans represent a virtually untapped resource for the discovery of novel bioactive compounds