Cytotoxic Natural Products from the Jurassic Relict Osmunda regalis L.

The Jurassic relict Royal fern, Osmunda regalis L., is widely distributed across temperate zones in the Northern and Southern hemispheres. Even though this species has been utilised for centuries as a medicinal plant, its phytochemical composition mainly remains unknown. As part of our ongoing research to identify potential lead compounds for future anticancer drugs, 17 natural products were characterised from the aerial parts of Osmunda regalis L. Fifteen of these compounds were identified in this species for the first time, including the six previously undescribed compounds kaempferol 3-O-(2''-O-(2'''-α-rhamnopyranosyl)-ß-glucopyranosyl)-ß-glucopyranoside, quercetin 3-O-(2''-O-(2'''-α-rhamnopyranosyl)-ß-glucopyranosyl)-ß-glucopyranoside, kaempferol 3-O-(2''-O-(2'''-α-rhamnopyranosyl-6'''-O-(E)-caffeoyl-)-ß-glucopyranosyl)-ß-glucopyranoside, 3-methoxy-5-hydroxy-4-olide, 4-hydroxy-3-(3'-hydroxy-4'-(hydroxyethyl)-oxotetrafuranone-5-methyl tetrahydropyranone, and 4-O-(5-hydroxy-4-oxohexanoyl) osmundalactone. The molecular structures were determined by combining several 1D and 2D NMR experiments, circular dichroism spectroscopy, and HRMS. Determination of cytotoxicity against AML MOLM-13, H9c2, and NRK cell lines showed that two isolated lactones exhibited significant cytotoxic activity.

Cytotoxic Natural Products Isolated from Cryptogramma crispa (L.) R. Br.

Parsley fern, Cryptogramma crispa, is a common fern in arctic-alpine regions, and even though this species has been known since ancient times and has been presumed to cause the poisoning of horses, its natural products have not previously been investigated. Here, we characterise 15 natural products isolated from the aerial parts of Cryptogramma crispa, including the previously undescribed compound 3-malonyl pteroside D. The structure determinations were based on several advanced 1D and 2D NMR spectroscopic techniques, Circular Dichroism spectroscopy and high-resolution mass spectrometry. The pteroside derivatives exhibited selective moderate cytotoxic activity against the acute myeloid leukaemia MOLM13 cell line and no cytotoxicity against the normal heart and kidney cell lines, suggesting that their potential anticancer effect should be further investigated.

Natural Products from Leaves of the Ancient Iranian Medicinal Plant Echium amoenum Fisch. & C. A. Mey.

For several millennia, leaves of Echium amoenum Fisch. & C. A. Mey., an important Iranian medicinal plant with nutritional value as nutraceutical, have been used as tea for the treatment of several conditions, including inflammation. The nutritional value of intake of E. amoenum tea has mainly been correlated to its rich content of mainly water-soluble antioxidants. Although the entire plant is utilized, only natural products of the flowers have previously been thoroughly investigated. The rare natural products bis(3-(3,4-dihydroxyphenyl)-1-methoxy-1-oxopropan-2-yl)-1-(3,4-dihydroxyphenyl)-6,7-dihydroxy-1,2-dihydronaphthalene-2,3-dicarboxylate, 4-Oxy-(E)-caffeoyl-2,3-dihydroxybutanoic acid methyl ester and 4-Oxy-(Z)-caffeoyl-2,3-dihydroxybutanoic acid methyl ester, in addition to the widely distributed compounds rosmarinic acid methyl ester and (E)-caffeic acid, were purified and characterized from leaves of Echium amoenum. The structures were determined by a combination of several 2D NMR spectroscopic techniques, circular dichroism spectroscopy and high-resolution mass spectrometry. The fact that bis(3-(3,4-dihydroxyphenyl)-1-methoxy-1-oxopropan-2-yl)-1-(3,4-dihydroxyphenyl)-6,7-dihydroxy-1,2-dihydronaphthalene-2,3-dicarboxylate belongs to a rare group of natural products which have previously been patented for their significant anti-inflammatory activity may rationalize the traditional treatment of inflammations with E. amoenum.

Phosphosite Analysis of the Cytomegaloviral mRNA Export Factor pUL69 Reveals Serines with Critical Importance for Recruitment of Cellular Proteins Pin1 and UAP56/URH49.

Human cytomegalovirus (HCMV) encodes the viral mRNA export factor pUL69, which facilitates the cytoplasmic accumulation of mRNA via interaction with the cellular RNA helicase UAP56 or URH49. We reported previously that pUL69 is phosphorylated by cellular CDKs and the viral CDK-like kinase pUL97. Here, we set out to identify phosphorylation sites within pUL69 and to characterize their importance. Mass spectrometry-based phosphosite mapping of pUL69 identified 10 serine/threonine residues as phosphoacceptors. Surprisingly, only a few of these sites localized to the N terminus of pUL69, which could be due to the presence of additional posttranslational modifications, like arginine methylation. As an alternative approach, pUL69 mutants with substitutions of putative phosphosites were analyzed by Phos-tag SDS-PAGE. This demonstrated that serines S46 and S49 serve as targets for phosphorylation by pUL97. Furthermore, we provide evidence that phosphorylation of these serines mediates cis/trans isomerization by the prolyl isomerase Pin1, thus forming a functional Pin1 binding motif. Surprisingly, while abrogation of the Pin1 motif did not affect the replication of recombinant cytomegaloviruses, mutation of serines next to the interaction site for UAP56/URH49 strongly decreased viral replication. This was correlated with a loss of UAP56/URH49 recruitment. Intriguingly, the critical serines S13 and S15 were located within a sequence resembling the UAP56 binding motif (UBM) of cellular mRNA adaptor proteins like REF and UIF. We propose that betaherpesviral mRNA export factors have evolved an extended UAP56/URH49 recognition sequence harboring phosphorylation sites to increase their binding affinities. This may serve as a strategy to successfully compete with cellular mRNA adaptor proteins for binding to UAP56/URH49.IMPORTANCE The multifunctional regulatory protein pUL69 of human cytomegalovirus acts as a viral RNA export factor with a critical role in efficient replication. Here, we identify serine/threonine phosphorylation sites for cellular and viral kinases within pUL69. We demonstrate that the pUL97/CDK phosphosites within alpha-helix 2 of pUL69 are crucial for its cis/trans isomerization by the cellular protein Pin1. Thus, we identified pUL69 as the first HCMV-encoded protein that is phosphorylated by cellular and viral serine/threonine kinases in order to serve as a substrate for Pin1. Furthermore, our study revealed that betaherpesviral mRNA export proteins contain extended binding motifs for the cellular mRNA adaptor proteins UAP56/URH49 harboring phosphorylated serines that are critical for efficient viral replication. Knowledge of the phosphorylation sites of pUL69 and the processes regulated by these posttranslational modifications is important in order to develop antiviral strategies based on a specific interference with pUL69 phosphorylation.

Acylated Flavone O-Glucuronides from the Aerial Parts of Nepeta curviflora.

Nepeta curviflora Boiss. (Syrian catnip) is native to the Middle East. This medicinal plant is commonly used against nervous disorders, rheumatic pains, and high blood pressure. Herbal infusions prepared from various Nepeta spp. are extensively consumed as functional food. However, limited information has been known about the phenolic constituents of Syrian catnip. In this study, two acylated flavone 7-O-glucuronides, apigenin 7-O-(2″-O-(2‴-(E-caffeoyl)-ß-glucuronopyranosyl)-ß-glucuronopyranoside) (1) and luteolin 7-O-(2″-O-(2‴-(E-caffeoyl)-ß-glucuronopyranosyl)-ß-glucuronopyranoside) (2), along with the known phenolic compounds rosmarinic acid, caffeic acid, apigenin, and apigenin 7-O-ß-glucopyranoside were isolated from the aerial parts of N. curviflora. The characterizations of these compounds were based on high-resolution mass spectrometry, UV, and extensive use of multidimensional NMR spectroscopy. The new compounds (1 and 2) were identified in the unmodified state and as dimethylesters.

The Prolyl Isomerase Pin1 Promotes the Herpesvirus-Induced Phosphorylation-Dependent Disassembly of the Nuclear Lamina Required for Nucleocytoplasmic Egress.

The nuclear lamina lines the inner nuclear membrane providing a structural framework for the nucleus. Cellular processes, such as nuclear envelope breakdown during mitosis or nuclear export of large ribonucleoprotein complexes, are functionally linked to the disassembly of the nuclear lamina. In general, lamina disassembly is mediated by phosphorylation, but the precise molecular mechanism is still not completely understood. Recently, we suggested a novel mechanism for lamina disassembly during the nuclear egress of herpesviral capsids which involves the cellular isomerase Pin1. In this study, we focused on mechanistic details of herpesviral nuclear replication to demonstrate the general importance of Pin1 for lamina disassembly. In particular, Ser22-specific lamin phosphorylation consistently generates a Pin1-binding motif in cells infected with human and animal alpha-, beta-, and gammaherpesviruses. Using nuclear magnetic resonance spectroscopy, we showed that binding of Pin1 to a synthetic lamin peptide induces its cis/trans isomerization in vitro. A detailed bioinformatic evaluation strongly suggests that this structural conversion induces large-scale secondary structural changes in the lamin N-terminus. Thus, we concluded that a Pin1-induced conformational change of lamins may represent the molecular trigger responsible for lamina disassembly. Consistent with this concept, pharmacological inhibition of Pin1 activity blocked lamina disassembly in herpesvirus-infected fibroblasts and consequently impaired virus replication. In addition, a phospho-mimetic Ser22Glu lamin mutant was still able to form a regular lamina structure and overexpression of a Ser22-phosphorylating kinase did not induce lamina disassembly in Pin1 knockout cells. Intriguingly, this was observed in absence of herpesvirus infection proposing a broader importance of Pin1 for lamina constitution. Thus, our results suggest a functional model of similar events leading to disassembly of the nuclear lamina in response to herpesviral or inherent cellular stimuli. In essence, Pin1 represents a regulatory effector of lamina disassembly that promotes the nuclear pore-independent egress of herpesviral capsids.

Influence of chain length on the activity of tripeptidomimetic antagonists for CXC chemokine receptor 4 (CXCR4).

Here we report a series of close analogues of our recently published scaffold-based tripeptidomimetic CXCR4 antagonists, containing positively charged guanidino groups in R1 and R2, and an aromatic group in R3. While contraction/elongation of the guanidine carrying side chains (R1 and R2) resulted in loss of activity, introduction of bromine in position 1 on the naphth-2-ylmethyl moiety (R3) resulted in an EC50 of 61µM (mixture of diastereoisomers) against wild-type CXCR4; thus, the antagonistic activity of these tripeptidomimetics seems to be amenable to optimization of the aromatic moiety. Moreover, for analogues carrying a naphth-2-ylmethyl substituent, we observed that a Pictet-Spengler like cyclization side reaction depended on the nature of the R1 substituent.

Non-Polar Natural Products from Bromelia laciniosa, Neoglaziovia variegata and Encholirium spectabile (Bromeliaceae).

Extensive regional droughts are already a major problem on all inhabited continents and severe regional droughts are expected to become an increasing and extended problem in the future. Consequently, extended use of available drought resistant food plants should be encouraged. Bromelia laciniosa , Neoglaziovia variegata and Encholirium spectabile are excellent candidates in that respect because they are established drought resistant edible plants from the semi-arid Caatinga region. From a food safety perspective, increased utilization of these plants would necessitate detailed knowledge about their chemical constituents. However, their chemical compositions have previously not been determined. For the first time, the non-polar constituents of B. laciniosa , N. variegata and E. spectabile have been identified. This is the first thorough report on natural products from N. variegata , E. spectabile , and B. laciniosa . Altogether, 20 non-polar natural products were characterized. The identifications were based on hyphenated gas chromatography-high resolution mass spectrometry (GC-HRMS) and supported by 1D and 2D Nuclear Magnetic Resonance (NMR) plant metabolomics.

pUL69 of Human Cytomegalovirus Recruits the Cellular Protein Arginine Methyltransferase 6 via a Domain That Is Crucial for mRNA Export and Efficient Viral Replication.

UNLABELLED: The regulatory protein pUL69 of human cytomegalovirus acts as a viral mRNA export factor, facilitating the cytoplasmic accumulation of unspliced RNA via interaction with the cellular mRNA export factor UAP56. Here we provide evidence for a posttranslational modification of pUL69 via arginine methylation within the functionally important N terminus. First, we demonstrated a specific immunoprecipitation of full-length pUL69 as well as pUL69aa1-146 by a mono/dimethylarginine-specific antibody. Second, we observed a specific electrophoretic mobility shift upon overexpression of the catalytically active protein arginine methyltransferase 6 (PRMT6). Third, a direct interaction of pUL69 and PRMT6 was confirmed by yeast two-hybrid and coimmunoprecipitation analyses. We mapped the PRMT6 interaction motif to the pUL69 N terminus and identified critical amino acids within the arginine-rich R1 box of pUL69 that were crucial for PRMT6 and/or UAP56 recruitment. In order to test the impact of putative methylation substrates on the functions of pUL69, we constructed various pUL69 derivatives harboring arginine-to-alanine substitutions and tested them for RNA export activity. Thus, we were able to discriminate between arginines within the R1 box of pUL69 that were crucial for UAP56/PRMT6-interaction and/or mRNA export activity. Remarkably, nuclear magnetic resonance (NMR) analyses revealed the same α-helical structures for pUL69 sequences encoding either the wild type R1/R2 boxes or a UAP56/PRMT6 binding-deficient derivative, thereby excluding the possibility that R/A amino acid substitutions within R1 affected the secondary structure of pUL69. We therefore conclude that the pUL69 N terminus is methylated by PRMT6 and that this critically affects the functions of pUL69 for efficient mRNA export and replication of human cytomegalovirus. IMPORTANCE: The UL69 protein of human cytomegalovirus is a multifunctional regulatory protein that acts as a viral RNA export factor with a critical role for efficient replication. Here, we demonstrate that pUL69 is posttranslationally modified via arginine methylation and that the protein methyltransferase PRMT6 mediates this modification. Furthermore, arginine residues with a crucial function for RNA export and for binding of the cellular RNA export factor UAP56 as well as PRMT6 were mapped within the arginine-rich R1 motif of pUL69. Importantly, we demonstrated that mutation of those arginines did not alter the secondary structure of R1, suggesting that they may serve as critical methylation substrates. In summary, our study reveals a novel posttranslational modification of pUL69 which has a significant impact on the function of this important viral regulatory protein. Since PRMTs appear to be amenable to selective inhibition by small molecules, this may constitute a novel target for antiviral therapy.

Growing with dinosaurs: natural products from the Cretaceous relict Metasequoia glyptostroboides Hu & Cheng-a molecular reservoir from the ancient world with potential in modern medicine.

After the sensational rediscovery of living exemplars of the Cretaceous relict Metasequoia glyptostroboides-a tree previously known exclusively from fossils from various locations in the northern hemisphere, there has been an increasing interest in discovery of novel natural products from this unique plant source. This article includes the first complete compilation of natural products reported from M. glyptostroboides during the entire period in which the tree has been investigated (1954-2014) with main focus on the compounds specific to this plant source. Studies on the biological activity of pure compounds and extracts derived from M. glyptostroboides are reviewed for the first time. The unique potential of M. glyptostroboides as a source of bioactive constituents is founded on the fact that the tree seems to have survived unchanged since the Cretaceous era. Since then, its molecular defense system has resisted the attacks of millions of generations of pathogens. In line with this, some recent landmarks in Metasequoia paleobotany are covered. Initial spectral analysis of recently discovered intact 53 million year old wood and amber of Metasequoia strongly indicate that the tree has remained unchanged for millions of years at the molecular level.

New Polyfunctional Phragmalin Limonoids from Neobeguea mahafalensis.

Neobeguea mahafalensis is used as a medicinal plant in Madagascar. A decoction of the stem bark of this species is reported to treat back pain. Recently, it was reported that a decoction of the root bark, containing two novel phragmalin limonoids as identified active constituents, exhibited an extraordinarily high potency and remarkably long duration in augmenting sexual activity in male rodents.From the dichloromethane extract of the root barks of N. mahafalensis, nine phragmalin limonoids were isolated, of which eight were novel compounds. The structures were established mainly by extensive use of 2D NMR spectroscopic techniques and high-resolution mass spectrometry. One of the new compounds named dodoguin displayed sleep-inducing activity in Swiss albino mice. The amount of 3-10 mg/kg of this compound induced sleep 18-22 min after its administration with a duration of 16-18 min.

HIV-1 p6 - a structured to flexible multifunctional membrane-interacting protein.

The human immunodeficiency virus type 1 (HIV-1) p6 protein has recently been recognized as a docking site for several cellular and viral binding partners and is important for the formation of infectious viruses. Most of its known functions are suggested to occur under hydrophobic conditions near the cytoplasmic membrane, where the protein is presumed to exist in its most structured state. Although p6 is involved in manifold specific interactions, the protein has previously been considered to possess a random structure in aqueous solution. We show that p6 exhibits a defined structure with N- and C-terminal helical domains, connected by a flexible hinge region in 100mM dodecylphosphocholine micelle solution at pH 7 devoid of any organic co-solvents, indicating that this is a genuine limiting structural feature of the molecule in a hydrophobic environment. Furthermore, we show that p6 directly interacts with a cytoplasmic model membrane through both N-terminal and C-terminal regions by use of surface plasmon resonance (SPR) spectroscopy. Phosphorylation of Ser-40 located in the center of the C-terminal α-helix does not alter the secondary structure of the protein but amplifies the interaction with membranes significantly, indicating that p6 binds to the polar head groups at the surface of the cytoplasmic membrane. The increased hydrophobic membrane interaction of p6(23-52) S40F correlated with the observed increased amount of the polyprotein Gag in the RIPA insoluble fraction when Ser40 of p6 was mutated with Phe indicating that p6 modulates the membrane interactions of HIV-1 Gag.

Influenza A virus protein PB1-F2 from different strains shows distinct structural signatures.

The proapoptotic influenza A virus PB1-F2 protein contributes to viral pathogenicity and is present in most human and avian influenza isolates. The structures of full-length PB1-F2 of the influenza strains Pandemic flu 2009 H1N1, 1918 Spanish flu H1N1, Bird flu H5N1 and H1N1 PR8, have been characterized by NMR and CD spectroscopy. The study was conducted using chemically synthesized full-length PB1-F2 protein and fragments thereof. The amino acid residues 30-70 of PR8 PB1-F2 were found to be responsible for amyloid formation of the protein, which could be assigned to formation of ß-sheet structures, although α-helices were the only structural features detected under conditions that mimic a membranous environment. At membranous conditions, in which the proteins are found in their most structured state, significant differences become apparent between the PB1-F2 variants investigated. In contrast to Pandemic flu 2009 H1N1 and PR8 PB1-F2, which exhibit a continuous extensive C-terminal α-helix, both Spanish flu H1N1 and Bird flu H5N1 PB1-F2 contain a loop region with residues 66-71 that divides the C-terminus into two shorter helices. The observed structural differences are located to the C-terminal ends of the proteins to which most of the known functions of these proteins have been assigned. A C-terminal helix-loop-helix motif might be a structural signature for PB1-F2 of the highly pathogenic influenza viruses as observed for 1918 Spanish flu H1N1 and Bird flu H5N1 PB1-F2. This signature could indicate the pathological nature of viruses emerging in the future and thus aid in the recognition of these viruses.

Design, synthesis, and biological evaluation of scaffold-based tripeptidomimetic antagonists for CXC chemokine receptor 4 (CXCR4).

Structure-activity relationship studies of the cyclopentapeptide CXCR4 antagonists (cyclo(-l-/d-Arg(1)-Arg(2)-2-Nal(3)-Gly(4)-d-Tyr(5)-)) suggest that the l-/d-Arg(1)-Arg(2)-2-Nal(3) tripeptide sequence contained within these cyclopentapeptides serves as a recognition motif for peptidic CXCR4 antagonists. Starting by dissecting the cyclopentapeptide structure and reintroducing cyclic constraints in a stepwise manner, we here report a novel class of scaffold-based tripeptidomimetic CXCR4 antagonists based on the d-Arg-Arg-2-Nal motif. Biological testing of the prototype compounds showed that they represent new peptidomimetic hits; importantly, the modular nature of the scaffold provides an interesting starting point for future ligand optimization.

Balticidins A-D, antifungal hassallidin-like lipopeptides from the Baltic Sea cyanobacterium Anabaena cylindrica Bio33.

Balticidins A-D (1-4), four new antifungal lipopeptides, were isolated from the laboratory-cultivated cyanobacterium Anabaena cylindrica strain Bio33 isolated from a water sample collected from the Baltic Sea, Rügen Island, Germany. Fractionation of the 50% aqueous MeOH extract was performed by bioassay-guided silica gel column chromatography followed by SPE and repeated reversed-phase HPLC. The main fraction containing the compounds exhibited a strong and specific antifungal activity with inhibition zones in an agar-diffusion assay from 21 to 32 mm against Candida albicans, Candida krusei, Candida maltosa, Aspergillus fumigatus, Microsporum gypseum, Mucor sp., and Microsporum canis. The structures were elucidated by multidimensional (1)H and (13)C NMR spectroscopy, HRESIMS, amino acid analysis, and sugar analysis. Spectroscopic data analysis afforded an unambiguous sequence of R.CHO(S1).CHOH.CONH-Thr(1)-Thr(2)-Thr(3)-HOTyr(4)-Dhb(5)-D-Gln(6)-Gly(7)-NMeThr(8)(S2)-L-Gln COOH(9), in which Dhb is dehydroaminobutyric acid, S1 is d(-)-arabinose-(3-1)-D-(+)-galacturonic acid, S2 is D-(+)-mannose, and R is the aliphatic residue -C13H26Cl or -C13H27. Besides NMeThr, D-allo-Thr, D-Thr, and L-Thr were identified, but the position of the enantiomers in the sequence is not clear. The four balticidins differ in their cyclic (2, 4)/linear (1, 3) core and the presence (1, 2)/absence (3, 4) of chlorine in the aliphatic unit.

HIV-1 p6-Another viral interaction partner to the host cellular protein cyclophilin A.

The 52-amino acid human immunodeficiency virus type 1 (HIV-1) p6 protein has previously been recognized as a docking site for several cellular and viral binding factors and is important for the formation of infectious viruses. A particular structural feature of p6 is the notably high relative content of proline residues, located at positions 5, 7, 10, 11, 24, 30, 37 and 49 in the sequence. Proline cis/trans isomerism was detected for all these proline residues to such an extent that more than 40% of all p6 molecules contain at least one proline in a cis conformation. 2D (1)H nuclear magnetic resonance analysis of full-length HIV-1 p6 and p6 peptides established that cyclophilin A (CypA) interacts as a peptidyl-prolyl cis/trans isomerase with all proline residues of p6. Only catalytic amounts of CypA were necessary for the interaction with p6 to occur, strongly suggesting that the observed interaction is highly relevant in vivo. In addition, surface plasmon resonance studies revealed binding of full-length p6 to CypA, and that this binding was significantly stronger than any of its N- or C-terminal peptides. This study demonstrates the first identification of an interaction between HIV-1 p6 and the host cellular protein CypA. The mode of interaction involves both transient enzyme-substrate interactions and a more stable binding. The binding motifs of p6 to Tsg-101, ALIX and Vpr coincide with binding regions and catalytic sites of p6 to CypA, suggesting a potential role of CypA in modulating functional interactions of HIV-1.

Novel GHB-derived natural products from European mistletoe (Viscum album).

CONTEXT: The European white-berry mistletoe [Viscum album L. (Loranthaceae)] is among the oldest known medicinal plants. At present the most important application of mistletoe extracts is in the treatment of cancer. However, natural products specific to mistletoe have rarely been encountered in the current literature. OBJECTIVE: To discover novel natural products specific to European mistletoe. MATERIALS AND METHODS: European mistletoe was extracted with methanol, purified to partition against diethyl ether and further purified with XAD-7 column chromatography. Pure compounds were separated by Sephadex column chromatography and preparative HPLC. The structures of the novel compounds were established using a combination of several 2D NMR spectroscopic techniques and mass spectrometry. RESULTS: A new type of natural product derived from the methyl ester of γ-hydroxybutyric acid (GHB) coupled to hydroxybenzoic acids, namely 3-(3'-carbomethoxypropyl) gallic acid and 3-(3'-carbomethoxypropyl)-7→3″-protocatechoyl galloate were characterized from European white-berry mistletoe. Condensation of the 3-hydroxyl of gallic acid with the 4-hydroxyl of GHB significantly reduced the radical scavenging properties of the former compound. DISCUSSION AND CONCLUSION: The characterized compounds define a novel group of natural products that may be of particular interest because it appears that the two new compounds are not closely related to any known natural product.

Nuclear import of isoforms of the cytomegalovirus kinase pUL97 is mediated by differential activity of NLS1 and NLS2 both acting through classical importin-α binding.

The multifunctional protein kinase pUL97 of human cytomegalovirus (HCMV) strongly determines the efficiency of virus replication. Previously, the existence of two pUL97 isoforms that arise from alternative translational initiation and show a predominant nuclear localization was described. Two bipartite nuclear localization sequences, NLS1 and NLS2, were identified in the N terminus of the large isoform, whilst the small isoform exclusively contained NLS2. The current study found the following: (i) pUL97 nuclear localization in HCMV-infected primary fibroblasts showed accumulations in virus replication centres and other nuclear sections; (ii) in a quantitative evaluation system for NLS activity, the large isoform showed higher efficiency of nuclear translocation than the small isoform; (iii) NLS1 was mapped to aa 6-35 and NLS2 to aa 190-213; (iv) using surface plasmon resonance spectroscopy, the binding of both NLS1 and NLS2 to human importin-α was demonstrated, stressing the importance of individual arginine residues in the bipartite consensus motifs; (v) nuclear magnetic resonance spectroscopy of pUL97 peptides confirmed an earlier statement about the functional requirement of NLS1 embedding into an intact α-helical structure; and (vi) a bioinformatics investigation of the solvent-accessible surface suggested a high accessibility of NLS1 and an isoform-specific, variable accessibility of NLS2 for interaction with importin-α. Thus, the nucleocytoplasmic transport mechanism of the isoforms appeared to be differentially regulated, and this may have consequences for isoform-dependent functions of pUL97 during virus replication.

A novel bicyclic lactone and other polyphenols from the commercially important vegetable Anthriscus cerefolium.

Garden chervil, Anthriscus cerefolium (L.) Hoffm. is an important herb commonly applied in Norwegian large-scale commercial kitchens. This species is a highly enriched source of phenolics, containing 1260 mg gallic acid equivalents (GAE) 100-1 g DM, however, the individual phenolic compounds have been scarcely characterized. Here we report on the qualitative and quantitative content of phenolics in garden chervil. The structure of the main phenolic compound was elucidated to be the previously undescribed compound 1,3-dicaffeoyl-5-malonyl-δ-quinide (1) by means of 1D- and 2D NMR and high-resolution mass spectrometry. The known flavones apigenin 7-O-ß-(2″-apiofuranosylglucopyranoside) (= apiin) (2), apigenin 7-(2″-apiosyl-6″-malonylglucoside) (3) and luteolin 7-glucoside (4) were also identified. Compound 3 is reported for the first time from this plant species. The main phenolic compound, 1,3-dicaffeoyl-5-malonyl-δ-quinide, exhibited moderate cytotoxicity towards acute monocytic leukaemia cells (MOLM-13) and rat kidney epithelial cells (NRK) with EC50 between 400 and 600 µM.

Highly conserved serine residue 40 in HIV-1 p6 regulates capsid processing and virus core assembly.

BACKGROUND: The HIV-1 p6 Gag protein regulates the final abscission step of nascent virions from the cell membrane by the action of two late assembly (L-) domains. Although p6 is located within one of the most polymorphic regions of the HIV-1 gag gene, the 52 amino acid peptide binds at least to two cellular budding factors (Tsg101 and ALIX), is a substrate for phosphorylation, ubiquitination, and sumoylation, and mediates the incorporation of the HIV-1 accessory protein Vpr into viral particles. As expected, known functional domains mostly overlap with several conserved residues in p6. In this study, we investigated the importance of the highly conserved serine residue at position 40, which until now has not been assigned to any known function of p6. RESULTS: Consistently with previous data, we found that mutation of Ser-40 has no effect on ALIX mediated rescue of HIV-1 L-domain mutants. However, the only feasible S40F mutation that preserves the overlapping pol open reading frame (ORF) reduces virus replication in T-cell lines and in human lymphocyte tissue cultivated ex vivo. Most intriguingly, L-domain mediated virus release is not dependent on the integrity of Ser-40. However, the S40F mutation significantly reduces the specific infectivity of released virions. Further, it was observed that mutation of Ser-40 selectively interferes with the cleavage between capsid (CA) and the spacer peptide SP1 in Gag, without affecting cleavage of other Gag products. This deficiency in processing of CA, in consequence, led to an irregular morphology of the virus core and the formation of an electron dense extra core structure. Moreover, the defects induced by the S40F mutation in p6 can be rescued by the A1V mutation in SP1 that generally enhances processing of the CA-SP1 cleavage site. CONCLUSIONS: Overall, these data support a so far unrecognized function of p6 mediated by Ser-40 that occurs independently of the L-domain function, but selectively affects CA maturation and virus core formation, and consequently the infectivity of released virions.