Virus-Induced Gene Silencing to Investigate Alkaloid Biosynthesis in Opium Poppy.

Virus-induced gene silencing (VIGS) enables the targeted silencing of genes in opium poppy (Papaver somniferum) and has been used extensively to determine or support the physiological functions of benzylisoquinoline alkaloid biosynthetic enzymes. Here we describe detailed protocols involved in the application of VIGS to investigate BIA metabolism in opium poppy.

Author Correction: Gene clustering and copy number variation in alkaloid metabolic pathways of opium poppy.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Gene clustering and copy number variation in alkaloid metabolic pathways of opium poppy.

Genes in plant secondary metabolic pathways enable biosynthesis of a range of medically and industrially important compounds, and are often clustered on chromosomes. Here, we study genomic clustering in the benzylisoquinoline alkaloid (BIA) pathway in opium poppy (Papaver somniferum), exploring relationships between gene expression, copy number variation, and metabolite production. We use Hi-C to improve the existing draft genome assembly, yielding chromosome-scale scaffolds that include 35 previously unanchored BIA genes. We find that co-expression of BIA genes increases within clusters and identify candidates with unknown function based on clustering and covariation in expression and alkaloid production. Copy number variation in critical BIA genes correlates with stark differences in alkaloid production, linking noscapine production with an 11-gene deletion, and increased thebaine/decreased morphine production with deletion of a T6ODM cluster. Our results show that the opium poppy genome is still dynamically evolving in ways that contribute to medically and industrially important phenotypes.

Purine Permease-Type Benzylisoquinoline Alkaloid Transporters in Opium Poppy.

Although opiate biosynthesis has been largely elucidated, and cell-to-cell transport has been long postulated, benzylisoquinoline alkaloid (BIA) transporters from opium poppy (Papaver somniferum) have not been reported. Investigation of a purine permease-type sequence within a recently discovered opiate biosynthetic gene cluster led to the discovery of a family of nine homologs designated as BIA uptake permeases (BUPs). Initial expression studies in engineered yeast hosting segments of the opiate pathway showed that six of the nine BUP homologs facilitated dramatic increases in alkaloid yields. Closer examination revealed the ability to uptake a variety of BIAs and certain pathway precursors (e.g. dopamine), with each BUP displaying a unique substrate acceptance profile. Improvements in uptake for yeast expressing specific BUPs versus those devoid of the heterologous transporters were high for early intermediates (300- and 25-fold for dopamine and norcoclaurine, respectively), central pathway metabolites [10-fold for (S)-reticuline], and end products (30-fold for codeine). A coculture of three yeast strains, each harboring a different consecutive segment of the opiate pathway and BUP1, was able to convert exogenous Levodopa to 3 ± 4 mg/L codeine via a 14-step bioconversion process involving over a dozen enzymes. BUP1 is highly expressed in opium poppy latex and is localized to the plasma membrane. The discovery of the BUP transporter family expands the role of purine permease-type transporters in specialized metabolism, and provides key insight into the cellular mechanisms involved in opiate alkaloid biosynthesis in opium poppy.

Neopinone isomerase is involved in codeine and morphine biosynthesis in opium poppy.

The isomerization of neopinone to codeinone is a critical step in the biosynthesis of opiate alkaloids in opium poppy. Previously assumed to be spontaneous, the process is in fact catalyzed enzymatically by neopinone isomerase (NISO). Without NISO the primary metabolic products in the plant, in engineered microbes and in vitro are neopine and neomorphine, which are structural isomers of codeine and morphine, respectively. Inclusion of NISO in yeast strains engineered to convert thebaine to natural or semisynthetic opiates dramatically enhances formation of the desired products at the expense of neopine and neomorphine accumulation. Along with thebaine synthase, NISO is the second member of the pathogenesis-related 10 (PR10) protein family recently implicated in the enzymatic catalysis of a presumed spontaneous conversion in morphine biosynthesis.

Evaluating the Functionality of Conjunctiva Using a Rabbit Dry Eye Model.

Purpose. To assess the conjunctival functionality in a rabbit dry eye (DE) model. Methods. Nictitating membrane, lacrimal and Harderian glands were surgically excised from male New Zealand white rabbits using minimally invasive surgery. Fluorescein/rose Bengal staining of ocular surface (OS) and Schirmer test were done before (BE) and after excision (AE). The expression of interleukin- (IL-) 1ß, tumor necrosis factor- (TNF-) α, and MUC5AC proteins were estimated by immunoblotting from conjunctival impression cytology specimens. MUC5AC mRNA was quantified as well. The effect of epithelial sodium channel (ENaC) blockers on tear production and potential differences (PD) of OS were assessed under anesthesia in rabbits with and without surgery. Results. Increase in corneal and conjunctival staining was observed 1 month AE compared to BE. Schirmer tests failed to show decrease in tear production. Elevated IL-1ß, and TNF-α, 1 month AE indicated inflammation. MUC5AC expression was elevated 1 month AE. ENaC blockers did not improve tear production in rabbit eyes AE but characteristic changes in PD were observed in rabbits with surgery. Conclusions. DE biomarkers are important tools for OS assessment and MUC5AC expression is elevated in rabbit DE. PD measurement revealed significant electrophysiological changes in rabbits with surgery.

Tear Production After Bilateral Main Lacrimal Gland Resection in Rabbits.

PURPOSE: This study reports tear compensation observed in rabbits with bilateral resection of main lacrimal gland (LG) and explored the potential mechanisms. METHODS: Dry eye conditions were created by resection of nictitating membrane (NM), Harderian gland (HG), and main LG in eight (16 eyes) male New Zealand White rabbits. In addition to Schirmer test, Periodic acid-Schiff (PAS) staining, and ocular surface staining with fluorescein and rose Bengal, conjunctival impression cytology was employed before and up to 4 months after excision (AE). Using reverse transcription-quantitative polymerase chain reaction (RT-qPCR), expression of inflammatory biomarkers (IL-1ß, TNF-α, and matrix metalloproteinase-9) were monitored. Further, involvement of ionic and water transporters were investigated in conjunctival epithelium. RESULTS: Significant dry eye phenotypes in rabbits were observed 1 month AE, which corroborated with elevated biomarker mRNA expression. However, Schirmer test score and goblet cell numbers never decreased AE in conjunctival epithelium. Moreover, ocular surface staining, and biomarker expression declined to baseline in over 4 months AE. No upregulation was observed of the following conjunctival ionic transporters: cystic fibrosis transmembrane conductance regulator, sodium potassium chloride cotransporters, sodium potassium ATPase, and epithelial sodium channels. Instead, aquaporin (AQP) 4 and AQP5 were upregulated. Immunolocalization and immune blotting of AQP4 was demonstrated in rabbit conjunctival epithelium. CONCLUSIONS: In the absence of NM, HG, and main LG in rabbits, tear secretion was not decreased and significant improvement of dry eye phenotypes observed with time AE. Conjunctival AQPs are possibly involved in a compensatory tear fluid production.

Purification of the therapeutic antibody trastuzumab from genetically modified plants using safflower Protein A-oleosin oilbody technology.

Production of therapeutic monoclonal antibodies using genetically modified plants may provide low cost, high scalability and product safety; however, antibody purification from plants presents a challenge due to the large quantities of biomass that need to be processed. Protein A column chromatography is widely used in the pharmaceutical industry for antibody purification, but its application is limited by cost, scalability and column fouling problems when purifying plant-derived antibodies. Protein A-oleosin oilbodies (Protein A-OB), expressed in transgenic safflower seeds, are relatively inexpensive to produce and provide a new approach for the capture of monoclonal antibodies from plants. When Protein A-OB is mixed with crude extracts from plants engineered to express therapeutic antibodies, the Protein A-OB captures the antibody in the oilbody phase while impurities remain in the aqueous phase. This is followed by repeated partitioning of oilbody phase against an aqueous phase via centrifugation to remove impurities before purified antibody is eluted from the oilbodies. We have developed this purification process to recover trastuzumab, an anti-HER2 monoclonal antibody used for therapy against specific breast-cancers that over express HER2 (human epidermal growth factor receptor 2), from transiently infected Nicotiana benthamiana. Protein A-OB overcomes the fouling problem associated with traditional Protein A chromatography, allowing for the development of an inexpensive, scalable and novel high-resolution method for the capture of antibodies based on simple mixing and phase separation.

NHE2X3 DKO mice exhibit gender-specific NHE8 compensation.

NHE8, the newest member of the sodium/hydrogen exchanger family, is expressed in the epithelial cells of the intestine and the kidney. Intestinal expression of NHE8 is significantly higher than that of NHE2 and NHE3 at a young age, suggesting that NHE8 is an important player for intestinal sodium absorption during early development. The current study was designed to explore if NHE8 plays a compensatory role for the loss of NHE2 and NHE3 function in NHE2X3 double-knockout (NHE2X3 DKO) mice. We further explored the regulatory mechanism(s) responsible for the change in NHE8 expression in NHE2X3 DKO mice. We found that >95% of NHE2X3 DKO mice survived through weanling. However, only 60% of male NHE2X3 DKO mice and 88% of female NHE2X3 DKO mice survived to 6 wk of life. We also found that the expression of NHE8 in wild-type female mice was higher compared with wild-type male mice after puberty. In NHE2X3 KDO mice, NHE8 expression was increased in females but not in males. Using Caco-2 cells as a model of the small intestine, we showed that testosterone inhibited endogenous NHE8 expression by reducing NHE8 mRNA synthesis, whereas estrogen had no effect on NHE8 expression. Thus our data show for the first time that intestinal NHE8 has a compensatory role in NHE2X3 DKO mice and this regulation is gender-dependent.

Tumor necrosis factor-{alpha} downregulates intestinal NHE8 expression by reducing basal promoter activity.

NHE8 transporter is a member of the sodium/hydrogen exchanger (NHE) family. This transporter protein is expressed at the apical membrane of epithelial cells of kidney and intestine and contributes to vectorial Na(+) transport in both tissues. Although NaCl absorption has been shown to be reduced in diarrhea associated with colitis and enteritis, little is known about the role of Na(+)/H(+) exchange and the involvement of NHE isoforms in the pathogenesis of inflammatory disorders and the mechanism of inflammation-associated diarrhea. This study investigated the role of NHE8 in the setting of inflammatory states. Jejunal mucosa was harvested from trinitrobenzene sulfonic acid (TNBS) colitis rats or lipopolysaccharide (LPS) rats for RNA extraction and brush-border membrane protein purification. The human NHE8 gene promoter was cloned from human genomic DNA and characterized in Caco-2 cells. The promoter was further used to study the mechanisms of TNF-alpha-mediated NHE8 expression downregulation in Caco-2 cells. Results from Western blot and real-time PCR indicated that NHE8 protein and mRNA were significantly reduced in TNBS rats and LPS rats. In Caco-2 cells, TNF-alpha produces similar reduction levels in the endogenous NHE8 mRNA expression observed in our in vivo studies. The downregulation of NHE8 expression mediated by TNF-alpha could be blocked by transcription inhibitor actinomycin D, suggesting the involvement of transcriptional regulation. Further studies indicated that the human NHE8 gene transcription could be activated by Sp3 transcriptional factor, and TNF-alpha inhibits human NHE8 expression by reducing Sp3 interaction at the minimal promoter region of the human NHE8 gene. In conclusion, our studies suggest that TNF-alpha decreases NHE8 expression in inflammation induced by TNBS and LPS, which may contribute to the diarrhea associated with inflammation.

Assembly of hepatitis delta virus: particle characterization, including the ability to infect primary human hepatocytes.

Efficient assembly of hepatitis delta virus (HDV) was achieved by cotransfection of Huh7 cells with two plasmids: one to provide expression of the large, middle, and small envelope proteins of hepatitis B virus (HBV), the natural helper of HDV, and another to initiate replication of the HDV RNA genome. HDV released into the media was assayed for HDV RNA and HBV envelope proteins and characterized by rate-zonal sedimentation, immunoaffinity purification, electron microscopy, and the ability to infect primary human hepatocytes. Among the novel findings were that (i) immunostaining for delta antigen 6 days after infection with 300 genome equivalents (GE) per cell showed only 1% of cells as infected, but this was increased to 16% when 5% polyethylene glycol was present during infection; (ii) uninfected cells did not differ from infected cells in terms of albumin accumulation or the presence of E-cadherin at cell junctions; and (iii) sensitive quantitative real-time PCR assays detected HDV replication even when the multiplicity of infection was 0.2 GE/cell. In the future, this HDV assembly and infection system can be further developed to better understand the mechanisms shared by HBV and HDV for attachment and entry into host cells.

Subcloning, localization, and expression of the rat intestinal sodium-hydrogen exchanger isoform 8.

Apically expressed intestinal and renal sodium-hydrogen exchangers (NHEs) play a major role in Na(+) absorption. Our previous studies on NHE ontogeny have shown that NHE-2 and NHE-3 are expressed at very low levels in young animals. Furthermore, single and/or double NHE-2 and NHE-3 knockout mice display no obvious abnormalities before weaning. These observations suggest that other transporter(s) may be involved in intestinal Na+ absorption during early life. The present studies were designed to clone the novel rat intestinal NHE-8 cDNA and to decipher the NHE-8 protein localization and gene expression pattern during different developmental stages. The rat NHE-8 cDNA has 2,160 bp and encodes a 575-amino acid protein. An antibody against NHE-8 protein was developed. Immunohistochemistry staining indicated apical localization of NHE-8 protein in rat intestinal epithelial cells. The apical localization of NHE-8 was also confirmed by its presence in brush-border membrane and its absence in basolateral membrane preparations. Northern blotting utilizing a NHE-8-specific probe demonstrated higher NHE-8 mRNA expression in young animals compared with adult animals. Western blot analysis revealed a similar pattern. Tissue distribution with multiple human tissue RNA blot showed that NHE-8 was expressed in multiple tissues including the gastrointestinal tract. In conclusion, we have cloned the full-length NHE-8 cDNA from rat intestine and further showed its apical localization in intestinal epithelial cells. We have also shown that NHE-8 gene expression and protein expression were regulated during ontogeny. Our data suggests that NHE-8 may play an important role in intestinal Na+ absorption during early life.

Effects of angiotensin II on NaPi-IIa co-transporter expression and activity in rat renal cortex.

The kidney plays a major role in reabsorption of phosphate with the majority occurring in the proximal tubule (PT). The type IIa sodium-phosphate co-transporter (NaPi-IIa) is the main player in PT. The purpose of current study was to determine the effect of angiotensin II (A-II) on membrane expression of NaPi-IIa in the rat renal cortex. A-II (500 ng/kg/min) was chronically infused into the Sprague-Dawley rats by miniosmotic pump for 7 days. The arterial pressure and circulating plasma A-II level along with urine output were markedly increased in A-II rats. There was diuresis but no natriuresis. The phosphate excretion increased sevenfold on day 4 and 5.7-fold on day 7. There was no change in Na-dependent Pi uptake in brush-border membrane (BBM) vesicles between A-II-treated group and control on day 4, however, there was a 43% increase on day 7. Western blot analysis of NaPi-IIa protein abundance showed a parallel pattern: no change after 4 days of treatment and a 48% increase after 7 days of treatment. However, Northern blot analysis of cortical RNA showed no change in NaPi-IIa mRNA abundance on day 7. A-II stimulation of Na/Pi co-transport activity is a result of increases in the expression of BBM NaPi-IIa protein level and that stimulation is most likely mediated by posttranscriptional mechanisms.

A motility revertant of the ndvB mutant of Bradyrhizobium japonicum.

A motility revertant of a Bradyrhizobium japonicum ndvB mutant was isolated and characterized. The ndvB mutants of B. japonicum have been reported to be osmotically sensitive, as well as defective in motility, periplasmic cyclic beta-(1-->3), (1-->6)-D-glucan synthesis, and symbiosis with soybean. The motility revertant was restored for osmotic tolerance but not for cyclic beta-glucan production or effective symbiosis. These results support our hypothesis that cyclic beta-glucans have an important role in symbiosis--the suppression of a plant defense response--in addition to their role in periplasmic osmoprotection.

Characterization of ndvD, the third gene involved in the synthesis of cyclic beta-(1 --> 3),(1 --> 6)-D-glucans in Bradyrhizobium japonicum.

Previously, we identified two genes in Bradyrhizobium japonicum (ndvB, ndvC) that are required for cyclic beta-(1 --> 3),(1 --> 6)-D-glucan synthesis and successful symbiotic interaction with soybean (Glycine max). In this study, we report a new open reading frame (ORF1) located in the intergenic region between ndvB and ndvC, which is essential for beta-glucan synthesis and effective nodulation of G. max. This new gene is designated ndvD (nodule development). The ndvD translation product has a predicted molecular mass of 26.4 kDa with one transmembrane domain. Genetic experiments involving gene deletion, Tn5 insertion, and gene complementation revealed that the mutation of ndvD generated pleiotropic phenotypes, including hypoosmotic sensitivity, reduced motility, and defects in conjugative gene transfer, in addition to symbiotic ineffectiveness. Although deficient in in vivo beta-glucan synthesis, membrane preparations from the ndvD mutant synthesized neutral beta-glucans in vitro. Therefore, ndvD does not appear to be a structural gene for beta-glucan synthesis. Our hypothesis for the mechanism of beta-(1 --> 3),(1 --> 6)-D-glucan synthesis is presented.