Ethylene response factor AcERF91 affects ascorbate metabolism via regulation of GDP-galactose phosphorylase encoding gene (AcGGP3) in kiwifruit.

Kiwifruit is known as 'the king of vitamin C' because of the high content of ascorbic acid (AsA) in the fruit. Deciphering the regulatory network and identification of the key regulators mediating AsA biosynthesis is vital for fruit nutrition and quality improvement. To date, however, the key transcription factors regulating AsA metabolism during kiwifruit developmental and ripening processes remains largely unknown. Here, we generated a putative transcriptional regulatory network mediating ascorbate metabolism by transcriptome co-expression analysis. Further studies identified an ethylene response factor AcERF91 from this regulatory network, which is highly co-expressed with a GDP-galactose phosphorylase encoding gene (AcGGP3) during fruit developmental and ripening processes. Through dual-luciferase reporter and yeast one-hybrid assays, it was shown that AcERF91 is able to bind and directly activate the activity of the AcGGP3 promoter. Furthermore, transient expression of AcERF91 in kiwifruit fruits resulted in a significant increase in AsA content and AcGGP3 transcript level, indicating a positive role of AcERF91 in controlling AsA accumulation via regulation of the expression of AcGGP3. Overall, our results provide a new insight into the regulation of AsA metabolism in kiwifruit.

The role of GDP-l-galactose phosphorylase in the control of ascorbate biosynthesis.

The enzymes involved in l-ascorbate biosynthesis in photosynthetic organisms (the Smirnoff-Wheeler [SW] pathway) are well established. Here, we analyzed their subcellular localizations and potential physical interactions and assessed their role in the control of ascorbate synthesis. Transient expression of C terminal-tagged fusions of SW genes in Nicotiana benthamiana and Arabidopsis thaliana mutants complemented with genomic constructs showed that while GDP-d-mannose epimerase is cytosolic, all the enzymes from GDP-d-mannose pyrophosphorylase (GMP) to l-galactose dehydrogenase (l-GalDH) show a dual cytosolic/nuclear localization. All transgenic lines expressing functional SW protein green fluorescent protein fusions driven by their endogenous promoters showed a high accumulation of the fusion proteins, with the exception of those lines expressing GDP-l-galactose phosphorylase (GGP) protein, which had very low abundance. Transient expression of individual or combinations of SW pathway enzymes in N. benthamiana only increased ascorbate concentration if GGP was included. Although we did not detect direct interaction between the different enzymes of the pathway using yeast-two hybrid analysis, consecutive SW enzymes, as well as the first and last enzymes (GMP and l-GalDH) associated in coimmunoprecipitation studies. This association was supported by gel filtration chromatography, showing the presence of SW proteins in high-molecular weight fractions. Finally, metabolic control analysis incorporating known kinetic characteristics showed that previously reported feedback repression at the GGP step, combined with its relatively low abundance, confers a high-flux control coefficient and rationalizes why manipulation of other enzymes has little effect on ascorbate concentration.

Association Between Galactose-Deficient IgA1 Derived From the Tonsils and Recurrence of IgA Nephropathy in Patients Who Underwent Kidney Transplantation.

Background: Recurrence of IgA nephropathy (IgAN) in the transplanted kidney is associated with graft survival, but no specific treatment is available. Tonsillectomy (TE) reportedly arrests the progression of IgAN in the native kidney. Thus, we conducted a single-center retrospective cohort study to evaluate the effect of TE prior to IgAN recurrence. Methods: Of the 36 patients with biopsy-proven IgAN who underwent kidney transplantation, 27 were included in this study. Nine patients underwent TE at 1 year after kidney transplantation (group 1), and the remaining 18 did not undergo TE (group 2). Results: The rate of histological IgAN recurrence was significantly lower in group 1 than in group 2 (11.1 vs. 55.6%, log-rank p = 0.046). In addition, half of the recurrent patients in group 2 exhibited active lesions, compared to none in group 1. Serum Gd-IgA1 levels decreased after TE in group 1, whereas they remained stable or increased slightly in group 2. In the recurrent cases, IgA and Gd-IgA1 were found in the germinal center in addition to the mantle zone of tonsils. Finally, mesangial IgA and Gd-IgA1 immunoreactivity was reduced after TE in some cases. Conclusion: Our data suggest that TE at 1 year after kidney transplantation might be associated with the reduced rate of histological IgAN recurrence. TE arrested or reduced serum Gd-IgA1 and mesangial Gd-IgA1 immunoreactivity. Therefore, we generated a hypothesis that serum Gd-IgA1 derived from the tonsils may play a pivotal role in the pathogenesis of IgAN. Based on these findings, we need to conduct verification in a prospective randomized controlled trial.

Electroacupuncture improves cognitive deficits and insulin resistance in an OLETF rat model of Al/D-gal induced aging model via the PI3K/Akt signaling pathway.

To investigate the effect of electroacupuncture (EA) on cognitive function and insulin resistance (IR) in an Al/D-gal-induced aging model for Alzheimer's disease (AD) using Ostuka Long-Evans Tokushima Fatty (OLETF) rats. The Al/D-gal-OLETF rats for AD were randomly divided into the EA and non-EA groups. Cognitive function was assessed using the Morris water maze (MWM). The morphology of the hippocampal neurons was observed using hematoxylin & eosin (H&E) staining. Aß and total Tau in the hippocampus and cerebrospinal fluid (CSF) were detected using western blotting (WB) and enzyme-linked immunosorbent assay (ELISA). Fasting blood glucose (FPG) was determined using the glucose oxidase method. Plasma fasting insulin (FINS), serum C-peptide (C-P), and CSF insulin were detected using ELISA. The expression of the genes and proteins in the PI3K signaling pathway was detected using quantitative real-time PCR and WB. After EA intervention, the hippocampal Aß and total Tau protein levels, body weight, FPG, FINS, and C-P were significantly decreased. The MWM showed that the percentage of time in the target quadrant of the EA group was elevated in the probe test. The protein levels of p-IRS1, p-IRS2, IDE, and p-GSK3ß were significantly increased, while p-PI3K-p85α and p-Akt were decreased. In conclusion, EA improves cognitive function and insulin resistance in rat models of AD. The PI3K/Akt signaling pathway is involved in those changes.

Clinical significance of serum and mesangial galactose-deficient IgA1 in patients with IgA nephropathy.

INTRODUCTION: Galactose-deficient IgA1 (Gd-IgA1) is a critical pathogenic factor for IgA nephropathy (IgAN), but its value as a disease-specific biomarker remains controversial. We aimed to clarify the clinical significance of Gd-IgA1 in patients with IgAN. METHODS: We retrospectively reviewed 111 patients who were diagnosed with IgAN based on the findings of renal biopsies (RB) at Showa University Hospital since 2007. Serum Gd-IgA1 (s-Gd-IgA1) at the time of RB was compared among 111 IgAN patients, 18 Henoch-Schönlein purpura nephritis (HSPN) patients, 29 lupus nephritis (LN) patients, 28 ANCA-associated vasculitis (AAV) patients, and 13 minimal change disease (MCD) patients using ELISA with an anti-human Gd-IgA1-specific monoclonal antibody (KM55). We also immunohistochemically stained paraffin-embedded sections for mesangial Gd-IgA1 (m-Gd-IgA1) deposition using KM55. RESULTS: Although levels of s-Gd-IgA1 were comparable among IgAN and HSPN, s-Gd-IgA1 levels were significantly elevated in patients with IgAN compared with LN, AAV and MCD (IgAN vs. HSPN, LN, AAV, and MCD: 16.2 ± 9.1 vs. 14.2 ± 10.8, p = 0.263; 12.7 ± 9.4, p = 0.008; 13.1 ± 7.3, p = 0.059; and 8.2 ± 4.8 µg/mL, p<0.001, respectively). Mesangial-Gd-IgA1 deposition was specifically detected in IgAN or HSPN. The increase in s-Gd-IgA1 significantly correlated with m-Gd-IgA1 positivity in patients with IgAN, and s-Gd-IgA1 elevation and m-Gd-IgA1 deposition were evident in patients with histopathologically advanced IgAN. Moreover, s-Gd-IgA1 levels were significantly higher in IgAN patients with glomerular sclerosis and tubulo-interstitial lesions. Mesangial-Gd-IgA1 intensity negatively correlated with eGFR in IgAN. Multivariate analysis selected s-Gd-IgA1 elevation as a significant risk factor for a 30%-reduction in eGFR in IgAN (HR, 1.37; 95% CI, 1.02-1.89; p = 0.038). CONCLUSIONS: Although IgAN and HSPN remain difficult to differentiate, s-Gd-IgA1 elevation and m-Gd-IgA1 deposition are reliable diagnostic factors that reflect IgAN severity. Serum-Gd-IgA1 could serve as a predictor of renal outcomes in IgAN. Thus, Gd-IgA1 could be significant biomarker for patients with IgAN.

Inflammation-induced Gro1 triggers senescence in neuronal progenitors: effects of estradiol.

BACKGROUND: Inflammation has been proposed to contribute to the decline in adult hippocampal neurogenesis. Proinflammatory cytokines activate transcription of chemokine growth-regulated oncogene α (Gro1) in human and murine hippocampal neuronal progenitor cells (NPC). The goal of this study was to investigate the effects of Gro1 on hippocampal neurogenesis in the presence of inflammation. METHODS: Human hippocampal NPC were transfected with lentivirus expressing Gro1, and murine NPC and hippocampal neuronal HT-22 cells were treated with Gro1 protein. A plasmid expressing mGro1 was electroporated in the hippocampus of newborn mice that were sacrificed 10 days later. Adult male and female mice were injected with lipopolysaccharide (LPS; 1 mg/kg, i.p in five daily injections) or normal saline. Adult male mice were implanted with pellets releasing 17-ß estradiol (E2; 2.5 mg/pellet, 41.666 µg/day release) or placebo for 6 weeks and challenged with LPS or normal saline as above. In both experiments, mice were sacrificed 3 h after the last injection. Hippocampal markers of neurogenesis were assessed in vitro and in vivo by Western blot, real-time PCR, and immunohisto/cytochemistry. RESULTS: Gro1 induced premature senescence in NPC and HT-22 cells, activating senescence-associated ß-galactosidase and the cell cycle inhibitor p16 and suppressing neuroblast proliferation and expression of doublecortin (DCX) and neuron-specific class III beta-tubulin (Tuj-1), both neuroblast markers, while promoting proliferation of neural glial antigen 2 (Ng2)-positive oligodendrocytes. Gro1 overexpression in the hippocampus of newborn mice resulted in decreased neuroblast development, as evidenced by decreased DCX expression and increased expression of platelet-derived growth factor α receptor (PDGFαR), a marker of oligodendrocyte precursors. In adult mice, Gro1 was induced in response to LPS treatment in male but not in female hippocampus, with a subsequent decrease in neurogenesis and activation of oligodendrocyte progenitors. No changes in neurogenesis were observed in females. Treatment with E2 blunted LPS-induced Gro1 in the male hippocampus. CONCLUSIONS: Inflammation-induced Gro1 triggers neuroblast senescence, thus suppressing new neuron development in the hippocampus. Sex-dependent differences in Gro1 response are attributed to estradiol, which blunts these changes, protecting the female hippocampus from the deleterious effects of inflammation-induced Gro1 on neurogenesis.

Neu5Gc and α1-3 GAL Xenoantigen Knockout Does Not Affect Glycemia Homeostasis and Insulin Secretion in Pigs.

Xenocell therapy from neonate or adult pig pancreatic islets is one of the most promising alternatives to allograft in type 1 diabetes for addressing organ shortage. In humans, however, natural and elicited antibodies specific for pig xenoantigens, α-(1,3)-galactose (GAL) and N-glycolylneuraminic acid (Neu5Gc), are likely to significantly contribute to xenoislet rejection. We obtained double-knockout (DKO) pigs lacking GAL and Neu5Gc. Because Neu5Gc-/- mice exhibit glycemic dysregulations and pancreatic ß-cell dysfunctions, we evaluated islet function and glucose metabolism regulation in DKO pigs. Isolation of islets from neonate piglets yielded identical islet equivalent quantities to quantities obtained from control wild-type pigs. In contrast to wild-type islets, DKO islets did not induce anti-Neu5Gc antibody when grafted in cytidine monophosphate-N-acetylneuraminic acid hydroxylase KO mice and exhibited in vitro normal insulin secretion stimulated by glucose and theophylline. Adult DKO pancreata showed no histological abnormalities, and immunostaining of insulin and glucagon was similar to that from wild-type pancreata. Blood glucose, insulin, C-peptide, the insulin-to-glucagon ratio, and HOMA-insulin resistance in fasted adult DKO pigs and blood glucose and C-peptide changes after intravenous glucose or insulin administration were similar to wild-type pigs. This first evaluation of glucose homeostasis in DKO pigs for two major xenoantigens paves the way to their use in (pre)clinical studies.

Improvement of betulinic acid biosynthesis in yeast employing multiple strategies.

BACKGROUND: Betulinic acid (BA) is a lupane-type triterpene which has been considered as a promising agent to cure melanoma with no side effects. Considering that BA is naturally produced in small quantities in plants, we previously reported the success in engineering its production in yeast. In the present study, we attempted to improve BA biosynthesis in yeast by the use of different strategies. RESULTS: We first isolated a gene encoding a lupeol C-28 oxidase (LO) from Betula platyphylla (designated as BPLO). BPLO showed a higher activity in BA biosynthesis compared to the previously reported LOs. In addition, two yeast platforms were compared for engineering the production of BA, which demonstrated that the WAT11 strain was better to host BA pathway than the CEN.PK strain. Based on the WAT11-chassiss, the Gal80p mutant was further constructed. The mutant produced 0.16 mg/L/OD600 of BA, which was 2.2 fold of that produced by the wild type strain (0.07 mg/L/OD600). CONCLUSIONS: This study reported our efforts to improve BA production in yeast employing multiple strategies, which included the identification of a novel LO enzyme with a higher activity in BA biosynthesis, the evaluation of two yeast strains for hosting the BA pathway, and the up-regulation of the expression of the BA pathway genes by managing yeast GAL gene regulon circuit.

Adaptively evolved yeast mutants on galactose show trade-offs in carbon utilization on glucose.

Adaptive evolution offers many opportunities in metabolic engineering; however, several constraints still exist as evolutionary trade-offs may impose collateral cost to obtain new traits. The application of adaptive evolution for strains development could be further improved by elucidating the molecular mechanisms. In this study, adaptively evolved yeast mutants with improved galactose utilization ability showed impaired glucose utilization. The molecular genetic basis of this trade-off was investigated using a systems biology approach. Transcriptional and metabolic changes resulting from the improvement of galactose utilization were found maintained during growth on glucose. Moreover, glucose repression related genes showed conserved expression patterns during growth on both sugars. Mutations in the RAS2 gene that were identified as beneficial for galactose utilization in evolved mutants exhibited significant correlation with attenuation of glucose utilization. These results indicate that antagonistic pleiotropy is the dominant mechanism in the observed trade-off, and it is likely realized by changes in glucose signaling.

Unusual galactofuranose modification of a capsule polysaccharide in the pathogenic yeast Cryptococcus neoformans.

Galactofuranose (Galf) is the five-membered ring form of galactose. Although it is absent from mammalian glycans, it occurs as a structural and antigenic component of important cell surface molecules in a variety of microbes, ranging from bacteria to parasites and fungi. One such organism is Cryptococcus neoformans, a pathogenic yeast that causes lethal meningoencephalitis in immunocompromised individuals, particularly AIDS patients. C. neoformans is unique among fungal pathogens in bearing a complex polysaccharide capsule, a critical virulence factor reported to include Galf. Notably, how Galf modification contributes to the structure and function of the cryptococcal capsule is not known. We have determined that Galf is ß1,2-linked to an unusual tetrasubstituted galactopyranose of the glucuronoxylomannogalactan (GXMGal) capsule polysaccharide. This discovery fills a longstanding gap in our understanding of a major polymer of the cryptococcal capsule. We also engineered a C. neoformans strain that lacks UDP-galactopyranose mutase; this enzyme forms UDP-Galf, the nucleotide sugar donor required for Galf addition. Mutase activity was required for the incorporation of Galf into glucuronoxylomannogalactan but was dispensable for vegetative growth, cell integrity, and virulence in a mouse model.

Recombinant jacalin-like plant lectins are produced at high levels in Nicotiana benthamiana and retain agglutination activity and sugar specificity.

The plant kingdom is an underexplored source of valuable proteins which, like plant lectins, display unique interacting specificities. Furthermore, plant protein diversity remains under-exploited due to the low availability and heterogeneity of native sources. All these hurdles could be overcome with recombinant production. A narrow phylogenetic gap between the native source and the recombinant platform is likely to facilitate proper protein processing and stability; therefore, the plant cell chassis should be specially suited for the recombinant production of many plant native proteins. This is illustrated herein with the recombinant production of two representatives of the plant jacalin-related lectin (JRLs) protein family in Nicotiana benthamiana using state-of-the-art magnICON technology. Mannose-specific Banlec JRL was produced at very high levels in leaves, reaching 1.0mg of purified protein per gram of fresh weight and showing strong agglutination activity. Galactose-specific jacalin JRL, with its complicated processing requirements, was also successfully produced in N. benthamiana at levels of 0.25 mg of purified protein per gram of fresh weight. Recombinant Jacalin (rJacalin) proved efficient in the purification of human IgA1, and was able to discriminate between plant-made and native IgA1 due to their differential glycosylation status. Together, these results show that the plant cell factory should be considered a primary option in the recombinant production of valuable plant proteins.

[Experimental study on A549 cell death mediated by xenoantigen α-gal 
in human serum].

BACKGROUND AND OBJECTIVE: The absence of α-gal in humans is caused by the inactivity of α-1,3GT gene. However, humans have pre-existing and abundant anti-gal antibodies. Xenotransplantation procedures have indicated the high potential of introducing α-1,3GT gene to synthesize α-gal for cancer gene therapy by mimicking hyper-acute rejection. The aim of this study is to construct a lung cancer A549 cell line that expressed α-gal, and to observe the antitumor mechanisms mediated by human serum. METHODS: A549 cells were transfected with pEGFP-N1-GT plasmids constructed in a previous study. A stable transgenic cell line, A549-GT, was then selected and cultivated. The biological characteristics of A549-GT cells, including morphology and proliferation, were examined. α-1,3GT mRNA expression was detected by RT-PCR. Direct immunofluorescence staining and flow cytometry (FCM) were used to analyze the synthesis of α-gal in A549-GT. The binding of human serum IgM and C3 with A549-GT were also detected. RESULTS: α-1,3GT mRNA was expressed in A549-GT. Direct immunofluorescence staining and FCM indicated a high and stable α-gal expression rate in A549-GT. Compared with parental A549 cells, the biological characteristics of A549-GT were unaltered. α-Gal expression was not detected in the human fetal lung fibroblast cell line MRC-5 even though A549-GT and its culture medium were cultivated with the enzyme. Immunofluorescence staining and FCM also indicated abundant binding between A549-GT treated with human serum and IgM/C3. CONCLUSIONS: α-Gal expression in tumor cells by gene transduction can induce complement-dependent cytototic antitumor effects.

Trade-off between responsiveness and noise suppression in biomolecular system responses to environmental cues.

When living systems detect changes in their external environment their response must be measured to balance the need to react appropriately with the need to remain stable, ignoring insignificant signals. Because this is a fundamental challenge of all biological systems that execute programs in response to stimuli, we developed a generalized time-frequency analysis (TFA) framework to systematically explore the dynamical properties of biomolecular networks. Using TFA, we focused on two well-characterized yeast gene regulatory networks responsive to carbon-source shifts and a mammalian innate immune regulatory network responsive to lipopolysaccharides (LPS). The networks are comprised of two different basic architectures. Dual positive and negative feedback loops make up the yeast galactose network; whereas overlapping positive and negative feed-forward loops are common to the yeast fatty-acid response network and the LPS-induced network of macrophages. TFA revealed remarkably distinct network behaviors in terms of trade-offs in responsiveness and noise suppression that are appropriately tuned to each biological response. The wild type galactose network was found to be highly responsive while the oleate network has greater noise suppression ability. The LPS network appeared more balanced, exhibiting less bias toward noise suppression or responsiveness. Exploration of the network parameter space exposed dramatic differences in system behaviors for each network. These studies highlight fundamental structural and dynamical principles that underlie each network, reveal constrained parameters of positive and negative feedback and feed-forward strengths that tune the networks appropriately for their respective biological roles, and demonstrate the general utility of the TFA approach for systems and synthetic biology.

Terminal N-linked galactose is the primary receptor for adeno-associated virus 9.

Sialylated glycans serve as cell surface attachment factors for a broad range of pathogens. We report an atypical example, where desialylation increases cell surface binding and infectivity of adeno-associated virus (AAV) serotype 9, a human parvovirus isolate. Enzymatic removal of sialic acid, but not heparan sulfate or chondroitin sulfate, increased AAV9 transduction regardless of cell type. Viral binding and transduction assays on mutant Chinese hamster ovary (CHO) cell lines defective in various stages of glycan chain synthesis revealed a potential role for core glycan residues under sialic acid in AAV9 transduction. Treatment with chemical inhibitors of glycosylation and competitive inhibition studies with different lectins suggest that N-linked glycans with terminal galactosyl residues facilitate cell surface binding and transduction by AAV9. In corollary, resialylation of galactosylated glycans on the sialic acid-deficient CHO Lec2 cell line with different sialyltransferases partially blocked AAV9 transduction. Quantitative analysis of AAV9 binding to parental, sialidase-treated or sialic acid-deficient mutant CHO cells revealed a 3-15-fold increase in relative binding potential of AAV9 particles upon desialylation. Finally, pretreatment of well differentiated human airway epithelial cultures and intranasal instillation of recombinant sialidase in murine airways enhanced transduction efficiency of AAV9 by >1 order of magnitude. Taken together, the studies described herein provide a molecular basis for low infectivity of AAV9 in vitro and a biochemical strategy to enhance gene transfer by AAV9 vectors in general.

Establishment of an mRNA gradient depends on the promoter: an investigation of polarity in gene expression.

We found six mRNA species specific to the galactose operon of Escherichia coli. Analyses of both ends of the mRNAs indicated that while the 5' ends are fixed at the promoter region, the 3' ends vary along the operon. The resulting gal mRNA map suggests generation of an mRNA concentration gradient that is higher in the promoter-proximal region and lower toward the distal region. Real-time RT-PCR analyses of the amount of each mRNA species confirmed the existence of the gradient. This gradient of mRNA concentration could serve as an underlying mechanism for the long known phenomenon "natural polarity." Further analyses of the 3' ends of the mRNAs showed that they are generated by either an unknown mRNA processing/transcription termination mechanism(s) or Rho-dependent intra-cistronic transcription termination. The results showed also that transcription from the P2 promoter can yield a more severe mRNA gradient than that from the P1 promoter, suggesting that the slope of the mRNA gradient depends on which promoter the transcription has initiated from. These results led us to suggest a novel gene regulation model in which transcription initiation is tightly coupled to mRNA processing and/or transcription termination.

Galactomutarotase and other galactose-related genes are rapidly induced by retinoic acid in human myeloid cells.

Aldose-1-epimerase (mutarotase) catalyzes the interconversion of alpha and beta hexoses, which is essential for normal carbohydrate metabolism and the production of complex oligosaccharides. Galactose mutarotase (GALM) has been well characterized at the protein level, but information is lacking on the regulation of GALM gene expression. We report herein that all-trans-retinoic acid (RA), an active metabolite of vitamin A that is known to induce myeloid lineage cell differentiation into macrophage-like cells, induces a rapid and robust regulation of GALM mRNA expression in human myeloid cells. all-trans-RA at a physiological concentration (20 nM), or Am580, a ligand selective for the nuclear retinoid receptor RARalpha, increased GALM mRNA in THP-1 cells, with significantly increased expression in 2 h, increasing further to an approximately 8-fold elevation after 6-40 h (P < 0.005). In contrast, tumor necrosis factor-alpha did not increase GALM mRNA expression, although it is capable of inducing cell differentiation. RA also increased GALM mRNA in U937 and HL-60 cells. The increase in GALM mRNA by RA was blocked by pretreating THP-1 cells with actinomycin D but not by cycloheximide. GALM protein and mutarotase activity were also increased time dependently in RA-treated THP-1 cells. In addition to GALM, several other genes in the biosynthetic pathway of galactosyl-containing complex oligosaccharides were more highly expressed in RA-treated THP-1 cells, including B4GALT5, ST3GAL3, ST6GALNAC5, and GALNAC4S-6ST. Thus, the results of this study identify RA as a significant regulator of GALM and other galactose-related genes in myeloid-monocytic cells, which could affect energy utilization and synthesis of cell-surface glycoproteins or glycolipids involved in cell motility, adhesion, and/or functional properties.

In vitro gene delivery to HepG2 cells using galactosylated 6-amino-6-deoxychitosan as a DNA carrier.

A chitosan derivative, 6-amino-6-deoxy chitosan (6ACT), was galactosylated and was investigated as a gene carrier. A series of galactose-modified 6ACT (Gal-6ACT) with degrees of substitution (d.s.) ranging from 3% to 50% per pyranose were prepared by reductive alkylation with lactose. DNA retardation assays showed that the electrostatic interaction between Gal-6ACT and plasmid DNA was not changed by galactose modification up to 50% per pyranose of 6ACT. Gal-6ACT with a d.s. of 38% was bound to galactose-recognizing lectin, RCA120. A significant increase in transfection efficiency for HepG2 cells was observed at degree of substitutions ranging from 18% to 50% and at N/P values ranging from 1.5 to 2.5. Under optimum conditions, Gal-6ACT showed about 10 times higher efficiency than 6ACT. However, a slight uptake by the galactose receptors on hepatocytes was observed by flow cytometric analysis. Moreover, Gal-6ACT with a d.s. of 38% mediated efficient gene transfer into both A549 and HeLa cells lacking the galactose receptor. These results suggest that the enhancement of transfection efficiency of Gal-6ACT was not due to the increase of receptor-mediated cellular uptake. In addition, the enhanced gene transfer efficiency was not specific to the galactose modification because the efficiency of glucose-modified 6ACT for HepG2 cells was similar as that of Gal-6ACT.

Improved understanding of gene expression regulation using systems biology.

This article reviews the current state of systems biology approaches, including the experimental tools used to generate 'omic' data and computational frameworks to interpret this data. Through illustrative examples, systems biology approaches to understand gene expression and gene expression regulation are discussed. Some of the challenges facing this field and the future opportunities in the systems biology era are highlighted.

Galactosylated ternary DNA/polyphosphoramidate nanoparticles mediate high gene transfection efficiency in hepatocytes.

Galactosylated polyphosphoramidates (Gal-PPAs) with different ligand substitution degrees (6.5%, 12.5% and 21.8%, respectively) were synthesized and evaluated as hepatocyte-targeted gene carriers. The in vitro cytotoxicity of Gal-PPA decreased significantly with an increase in galactose substitution degree. The affinity of Gal-PPA/DNA nanoparticles to galactose-recognizing lectin increased with galactose substitution degree. However, decreased transfection efficiency was observed for these galactosylated PPAs in HepG2 cells. Based on the results of gel retardation and polyanion competition assays, we hypothesized that the reduced transfection efficiency of Gal-PPA/DNA nanoparticles was due to their decreased DNA-binding capacity and decreased particle stability. We therefore prepared nanoparticles by precondensing DNA with PPA at a charge ratio of 0.5, yielding nanoparticles with negative surface charge, followed by coating with Gal-PPA, resulting in a Gal-PPA/ DNA/PPA ternary complex. Such a ternary nanoparticle formulation led to significant size reduction in comparison with binary nanoparticles, particularly at low N/P ratios (2 to 5). In HepG2 cells and primary rat hepatocytes, and at low N/P ratios (2 to 5), transfection efficiency mediated by ternary nanoparticles prepared with 6.5% Gal-PPA was 6-7200 times higher than PPA-DPA/DNA nanoparticles. Transgene expression increased slightly at higher N/P ratios in HepG2 cells and reached a plateau at N/P ratios between 5 and 10 for primary rat hepatocytes. Such an enhancement effect was not observed in HeLa cells that lack of asialoglycoprotein receptor (ASGPR). Nevertheless, transfection efficiency of ternary particles decreased dramatically, presumably due to the decreased DNA binding capacity and particle stability, as PPA galactosylation degree increased. This highlights the importance of optimizing ligand conjugation degree for PPA gene carrier.