Sheng-Xiong Huang

Decoding Human Cytomegalovirus

Dissecting HCMV Gene Expression Most of us are infected with human cytomegalovirus (HCMV), but severe disease is almost always limited to immunocompromised individuals or newborn infants. The virus has a relatively large (∼240 kb) DNA genome and shows a complex pattern of gene transcription, hinting at a complex regulatory and coding capacity. Stern-Ginossar et al. (p. 1088) mapped ribosome positions on HCMV transcripts during the course of viral infection of human fibroblast cells. The data suggest the presence of novel open reading frames (ORFs) lying within existing ORFs; very short ORFs upstream of canonical ORFs; ORFs antisense to canonical ORFs; and short, conserved ORFs encoded by long RNAs. Select ORFs were translated, dramatically expanding the coding capacity of the HCMV genome. A closer look at the human cytomegalovirus genome uncovers many new open reading frames. The human cytomegalovirus (HCMV) genome was sequenced 20 years ago. However, like those of other complex viruses, our understanding of its protein coding potential is far from complete. We used ribosome profiling and transcript analysis to experimentally define the HCMV translation products and follow their temporal expression. We identified hundreds of previously unidentified open reading frames and confirmed a fraction by means of mass spectrometry. We found that regulated use of alternative transcript start sites plays a broad role in enabling tight temporal control of HCMV protein expression and allowing multiple distinct polypeptides to be generated from a single genomic locus. Our results reveal an unanticipated complexity to the HCMV coding capacity and illustrate the role of regulated changes in transcript start sites in generating this complexity.

Diterpenoids from Isodon species and their biological activities

Isodon species (Labiatae) are widely distributed plants, many of which are used in folk medicine. Over the past twenty years, they have received considerable phytochemical and biological attention. Thestructures of their many diterpenoids constituents, especially those with an ent-kaurane skeleton, have been elucidated. The significant phytochemical and pharmacological diterpenoids form the subject of this review. There are 290 references.

Global mapping of translation initiation sites in mammalian cells at single-nucleotide resolution

Understanding translational control in gene expression relies on precise and comprehensive determination of translation initiation sites (TIS) across the entire transcriptome. The recently developed ribosome-profiling technique enables global translation analysis, providing a wealth of information about both the position and the density of ribosomes on mRNAs. Here we present an approach, global translation initiation sequencing, applying in parallel the ribosome E-site translation inhibitors lactimidomycin and cycloheximide to achieve simultaneous detection of both initiation and elongation events on a genome-wide scale. This approach provides a view of alternative translation initiation in mammalian cells with single-nucleotide resolution. Systemic analysis of TIS positions supports the ribosome linear-scanning mechanism in TIS selection. The alternative TIS positions and the associated ORFs identified by global translation initiation sequencing are conserved between human and mouse cells, implying physiological significance of alternative translation. Our study establishes a practical platform for uncovering the hidden coding potential of the transcriptome and offers a greater understanding of the complexity of translation initiation.

Genome Sequence of the Milbemycin-Producing Bacterium Streptomyces bingchenggensis

Streptomyces bingchenggensis is a soil-dwelling bacterium producing the commercially important anthelmintic macrolide milbemycins. Besides milbemycins, the insecticidal polyether antibiotic nanchangmycin and some other antibiotics have also been isolated from this strain. Here we report the complete genome sequence of S. bingchenggensis. The availability of the genome sequence of S. bingchenggensis should enable us to understand the biosynthesis of these structurally intricate antibiotics better and facilitate rational improvement of this strain to increase their titers.

Bacterial Diterpene Synthases: New Opportunities for Mechanistic Enzymology and Engineered Biosynthesis

Diterpenoid biosynthesis has been extensively studied in plants and fungi, yet cloning and engineering diterpenoid pathways in these organisms remain challenging. Bacteria are emerging as prolific producers of diterpenoid natural products, and bacterial diterpene synthases are poised to make significant contributions to our understanding of terpenoid biosynthesis. Here we will first survey diterpenoid natural products of bacterial origin and briefly review their biosynthesis with emphasis on diterpene synthases (DTSs) that channel geranylgeranyl diphosphate to various diterpenoid scaffolds. We will then highlight differences of DTSs of bacterial and higher organism origins and discuss the challenges in discovering novel bacterial DTSs. We will conclude by discussing new opportunities for DTS mechanistic enzymology and applications of bacterial DTS in biocatalysis and metabolic pathway engineering.

Bioassay-Guided Isolation of Xanthones and Polycyclic Prenylated Acylphloroglucinols from Garcinia oblongifolia

Bioassay-guided fractionation of the acetone extract of the bark of Garcinia oblongifolia has resulted in the isolation of three new xanthones, oblongixanthones A-C (1-3), three new polyprenylated benzoylphloroglucinols, oblongifolins E-G (4-6), and 12 known compounds. Oblongifolins I (5) and J (6) are the first natural products that have similar structural features to those of two known oxidation products of garcinol. The structures of the new compounds 1-6 were characterized by spectroscopic data interpretation. All isolates were assayed for their apoptosis-inducing effects against HeLa-C3 cells. Oblongifolin C (16) was found to be the most potent apoptotic inducer of the compounds evaluated.

Compounds from Kadsura heteroclita and related anti-HIV activity.

Phytochemical investigation of the stems of Kadsura heteroclita led to isolation of 16 compounds, including the triterpenoid named longipedlactone J (2), and two dibenzocyclooctadiene type lignans named heteroclitin I and J (3, 4). Compounds 8-10, 14, and 15 were weakly active as anti-HIV agents, whereas compounds 6 and 12 exhibited moderate anti-HIV activity with EC50 values of 1.6 microg/mL, and 1.4 microg/mL, therapeutic index (TI) values of 52.9, and 65.9, respectively. Their structures were established by spectroscopic methods, including application of 2D NMR techniques and CD spectra.

Bioactive Acylphloroglucinols with Adamantyl Skeleton from Hypericum sampsonii

Hyperisampsins A-D (1-4), with tetracyclo[6.3.1.1(3,10).0(3,7)]tridecane skeletons and seven biogenetically related congeners (5-11), were isolated from Hypericum sampsonii. Their structures were elucidated by comprehensive spectroscopic techniques. The absolute configuration of 1 was established by ECD calculations, and those of 5 and 9 were confirmed by single X-ray crystallographic analyses. Hyperisampsins A and D showed potent anti-HIV activities with EC50 of 2.97 and 0.97 μM and selectivity index of 4.80 and 7.70, respectively.

Comparative analysis of the biosynthetic gene clusters and pathways for three structurally related antitumor antibiotics: bleomycin, tallysomycin, and zorbamycin.

The biosynthetic gene clusters for the glycopeptide antitumor antibiotics bleomycin (BLM), tallysomycin (TLM), and zorbamycin (ZBM) have been recently cloned and characterized from Streptomyces verticillus ATCC15003, Streptoalloteichus hindustanus E465-94 ATCC31158, and Streptomyces flavoviridis ATCC21892, respectively. The striking similarities and differences among the biosynthetic gene clusters for the three structurally related glycopeptide antitumor antibiotics prompted us to compare and contrast their respective biosynthetic pathways and to investigate various enzymatic elements. The presence of different numbers of isolated nonribosomal peptide synthetase (NRPS) domains in all three clusters does not result in major structural differences of the respective compounds. The seemingly identical domain organization of the NRPS modules responsible for heterocycle formation, on the other hand, is contrasted by the biosynthesis of two different structural entities, bithiazole and thiazolinyl-thiazole, for BLM/TLM and ZBM, respectively. Variations in sugar biosynthesis apparently dictate the glycosylation patterns distinct for each of the BLM, TLM, and ZBM glycopeptide scaffolds. These observations demonstrate natures ingenuity and flexibility in achieving structural differences and similarities via various mechanisms and will surely inspire combinatorial biosynthesis efforts to expand on natural product structural diversity.

Bioassay and Ultraperformance Liquid Chromatography/Mass Spectrometry Guided Isolation of Apoptosis-Inducing Benzophenones and Xanthone from the Pericarp of Garcinia yunnanensis Hu

Bioassay and ultraperformance liquid chromatography/photodiode array/mass spectrometry (UPLC/PDA/MS) guided isolation of the apoptosis-inducing active metabolites on HeLa-C3 cells from the pericarp of Garcinia yunnanensis (Guttiferae) yielded five active compounds, including the new garciyunnanins A (1) and B (2). The structures of the compounds were elucidated by comprehensive nuclear magnetic resonance and mass spectrometry analysis. Garciyunnanin B (2), featured with a natural tetracyclic xanthone skeleton derived from a polyisoprenylated benzophenone, is structurally interesting since it can be seen as an evidence of the previously described cyclization of garcinol by 2,2-diphenyl-1-picrylhydrazyl (DPPH). Garciyunnanin A (1) contains a 3-monohydroxy benzophenone skeleton, which is rarely found in Garcinia species. Both new compounds induce HeLa-C3 cells into apoptosis after 72 h of incubation at 15 microM. It is noteworthy that oblongifolin C (4), the major constituent of this plant, has proved to be the most active one among the isolates for inducing apoptotic cell death in cervical cancer derived HeLa-C3 sensor cells.