Recycling of the major thylakoid lipid MGDG and its role in lipid homeostasis in Chlamydomonas reinhardtii.

Monogalactosyldiacylglycerol (MGDG), the most abundant lipid in thylakoid membranes, is involved in photosynthesis and chloroplast development. MGDG lipase has an important role in lipid remodeling in Chlamydomonas reinhardtii. However, the process related to turnover of the lysogalactolipid that results from MGDG degradation, monogalactosylmonoacylglycerol (MGMG), remains to be clarified. Here we identified a homolog of Arabidopsis thaliana lysophosphatidylcholine acyltransferase (LPCAT) and characterized two independent knockdown (KD) alleles in C. reinhardtii. The enzyme designated as C. reinhardtiiLysolipid Acyltransferase 1 (CrLAT1) has a conserved membrane-bound O-acyl transferase domain. LPCAT from Arabidopsis has a key role in deacylation of phosphatidylcholine (PC). Chlamydomonas reinhardtii, however, lacks PC, and thus we hypothesized that CrLAT1 has some other important function in major lipid flow in this organism. In the CrLAT1 KD mutants, the amount of MGMG was increased, but triacylglycerols (TAGs) were decreased. The proportion of more saturated 18:1 (9) MGDG was lower in the KD mutants than in their parental strain, CC-4533. In contrast, the proportion of MGMG has decreased in the CrLAT1 overexpression (OE) mutants, and the proportion of 18:1 (9) MGDG was higher in the OE mutants than in the empty vector control cells. Thus, CrLAT1 is involved in the recycling of MGDG in the chloroplast and maintains lipid homeostasis in C. reinhardtii.

4'-Nitrobiphenyl thioglucoside as the Smallest, fluorescent photosensitizer with cancer targeting ligand.

We have previously developed a glucose-linked biphenyl photosensitizer that can pass through glucose transporters, aiming for cancer-selective photodynamic therapy (PDT). Its small size (MW: 435) will allow oral administration and a fast clearance avoiding photosensitivity. However, its fluorescence efficiency was quite low, causing difficulty in monitoring cellular uptake. We thus synthesized a series of monosaccharide-linked biphenyl derivatives with a sulfur atom replacing an oxygen atom, in search of a photosensitizer with a brighter fluorescence. Among them, 4'-nitrobiphenyl thioglucoside showed a fluorescence emission extending to near infra-red region with a strength three times greater than that of the previous compound. This compound was found to have a higher 1O2-producing efficiency (ΦΔ: 0.75) than the previous compound (ΦΔ: 0.65). The thioglucoside indicated a significant photodamaging effect (IC50: 250 µM) against cancer cells. Although the galactose and mannose analogs exerted similar photodamaging effects, they were moderately toxic in the dark at a concentration of 300 µM. The thioglucoside and thiomannoside were at least partially uptaken through glucose transporters as demonstrated by inhibition with cytochalasin B, whereas no inhibition was observed for the galactoside. The behavior of d-glucose toward the cellular uptakes of these photosensitizers was bipolar: inhibitory at a low concentration and recovery or acceleratory at a higher concentration. These results indicate that 4'-nitrobiphenyl thioglucoside is the smallest (MW: 393) cancer-targeting photosensitizer with a trackable fluorescence property.

Affinity Control of Monosaccharide Conjugated Peptides against Lectins with a Set of Amino Acid Substitutions on α-Helical Structures.

Saccharides are well-known to play important roles in various biological events through specific interactions with target molecules such as carbohydrate-binding proteins (so-called lectins). Although characterization and identification of lectin molecules with saccharides are essential to understand biological events, they are still difficult due to weak interactions of saccharides, especially with monosaccharides. Herein, we demonstrate enhancement and control of monosaccharide affinity toward lectin proteins using chemical conjugation of monosaccharides with structurally regulated peptide and amino acid substitution. Thermodynamic analyses of the interactions by isothermal calorimetry measurements were performed to characterize the interactions between monosaccharide-conjugated peptide and the lectin molecules in detail. Conjugation with α-helical 16-mer short peptides drastically enhanced the affinity to lectins as compared with peptides with random coil structures, indicating that the α-helical peptide-based scaffold cooperatively interacted with lectins through additional interactions by suitable amino acids. Furthermore, suitable arrangement of the amino acids surrounding the monosaccharides on the α-helix afforded the conjugated peptides with varied affinities for two types of lectins. Our results indicate that the affinity of monosaccharide-conjugated peptides toward lectins is generally designable by appropriate conjugation of a simple monosaccharide with designed peptides, leading to the construction of a monosaccharide-modified peptide microarray toward high-throughput identification and/or screening of lectins in various biological events.

Photo effect on the CD1d-binding ability of azobenzene-attached analogues of α-GalCer.

α-Galactosylceramide (α-GalCer) is recognized by the CD1d proteins on antigen-presenting cells at the ceramide moiety and the galactose moiety is presented to iNKT cells, which stimulates the immune responses. However, the immune suppression by repeated injections of α-GalCer has discouraged its development as an anti-cancer agent. To overcome the shortcoming by spatiotemporal restriction of its exposure, we synthesized the photochromic azobenzene-incorporated analogues and tested the photo-immunoregulation effect in its binding to CD1d. FACS analyses indicated that some of these analogues enhanced the affinity to CD1d on photo-irradiation by about 20%. A docking simulation suggests that the photochromic molecule should be bulkier for a clearer discrimination between on and off states.

pH-Dependent Switching of Base Pairs Using Artificial Nucleobases with Carboxyl Groups.

In this study, we report the synthesis of modified oligonucleotides consisting of benzoic acid or isophthalic acid residues as new nucleobases. As evaluated by UV thermal denaturation analysis at different pH conditions (5.0, 6.0, 7.0, and 8.0), these modified oligonucleotides exhibited pH-dependent recognition of natural nucleobases and one is first found to be capable of base pair switching in response to a pH change. The isophthalic acid residue incorporated into the oligonucleotide on a d-threoninol backbone could preferentially bind with adenine but with guanine in response to a change in the pH conditions from pH 5 to pH 7 (or 8) without significant difference in duplex stability. These findings would be valuable for further developing pH-responsive DNA-based molecular devices.

Water-Soluble Glucosyl Pyrene Photosensitizers: An Intramolecularly Synthesized 2- C-Glucoside and an O-Glucoside.

Prevalent photosensitizing agents for photodynamic therapy (PDT) suffer from their relatively large molecular weights causing photodermatosis. In this regard, low molecular weight pyrene could be an efficient photosensitizer except for its extreme hydrophobicity. To tackle the insolubility of pyrene, we synthesized 1-carboxypyren-2-yl C-glucoside 4 by a tethered C-glucosylation and 1-pyrenylmethyl O-glucoside 5 by a simple O-glucosylation. Compounds 4 and 5 showed modest water solubilities of 72 and 47 µg/mL, respectively. Whereas compound 4 partially underwent a cyclization reaction at pH 3 to give the corresponding δ-valerolactone 15b in 31% yield after 24 h, it is stable at pH 5-9 for at least a week. The 1O2-producing photosensitizabilities of 4 and 5 were sufficient to apply to PDT. Although compound 5 was uptaken by HeLa cells and showed a good PDT activity, compound 4 showed neither a sufficient cell uptake nor PDT effect. The binding modes of compounds 4 and 5 to concanavalin A were specific and unspecific, respectively. These results demonstrate that compounds 4 and 5 are within a pharmacologically acceptable range as oral drugs and could be a fluorescence imaging probe for α-glucose/mannose receptors and a photosensitizing agent for PDT, respectively.

Synthesis of and triplex formation in oligonucleotides containing 2'-deoxy-6-thioxanthosine.

This study aimed to synthesize triplex-forming oligonucleotides (TFOs) containing 2'-deoxy-6-thioxanthosine (s6X) and 2'-deoxy-6-thioguanosine (s6Gs) residues and examined their triplex-forming ability. Consecutive arrangement of s6X and s6Gs residues increased the triplex-forming ability of the oligonucleotides more than 50 times, compared with the unmodified TFOs. Moreover, the stability of triplex containing a mismatched pair was much lower than that of the full-matched triplex, though s6X could form a s6X-GC mismatched pair via tautomerization of s6X. The present results reveal excellent properties of modified TFOs containing s6Xs and s6Gs residues, which may be harnessed in gene therapy and DNA nanotechnology.

Coating lanthanide nanoparticles with carbohydrate ligands elicits affinity for HeLa and RAW264.7 cells, enhancing their photodamaging effect.

Lanthanide nanoparticles (LNPs) conjugated with monosaccharides were synthesized as a photon energy-upconverting nanodevice with affinity to cancer cells. The conjugates were designed to selectively damage the cancer cells containing protoporphyrin IX, a photosensitizer endogenously synthesized from priorly administrated 5-aminolevlunic acid (ALA), by a highly tissue-penetrative near-infrared (NIR) irradiation. First of all, the affinities of monosaccharides toward cells (HeLa, RAW264.7, and MKN45) were assessed by a novel cell aggregation assay with trivalent monosaccharide-citric acid conjugates. As a result, HeLa exhibited high affinity for glucose, while RAW264.7 for glucose, galactose, mannose, and fucose. A similar cell-monosaccharide affinity was microscopically observed when the cells were mixed with monosaccharide-LNP conjugates and rinsed, in which the high affinity LNP probes luminesced on the cells. The high affinity monosaccharide-LNPs showed greater photodamaging effects than the unmodified LNP toward the corresponding cells, when the cells were pretreated with ALA and irradiated by NIR. This study demonstrates that carbohydrates can be used as selective ligands for cancer cells in a photodynamic therapy with LNP.

Synthesis and triplex-forming properties of oligonucleotides capable of recognizing corresponding DNA duplexes containing four base pairs.

A triplex-forming oligonucleotide (TFO) could be a useful molecular tool for gene therapy and specific gene modification. However, unmodified TFOs have two serious drawbacks: low binding affinities and high sequence-dependencies. In this paper, we propose a new strategy that uses a new set of modified nucleobases for four-base recognition of TFOs, and thereby overcome these two drawbacks. TFOs containing a 2'-deoxy-4N-(2-guanidoethyl)-5-methylcytidine (d(g)C) residue for a C-G base pair have higher binding and base recognition abilities than those containing 2'-OMe-4N-(2-guanidoethyl)-5-methylcytidine (2'-OMe (g)C), 2'-OMe-4N-(2-guanidoethyl)-5-methyl-2-thiocytidine (2'-OMe (g)Cs), d(g)C and 4S-(2-guanidoethyl)-4-thiothymidine ((gs)T). Further, we observed that N-acetyl-2,7-diamino-1,8-naphtyridine ((DA)Nac) has a higher binding and base recognition abilities for a T-A base pair compared with that of dG and the other DNA derivatives. On the basis of this knowledge, we successfully synthesized a fully modified TFO containing (DA)Nac, d(g)C, 2'-OMe-2-thiothymidine (2'-OMe (s)T) and 2'-OMe-8-thioxoadenosine (2'-OMe (s)A) with high binding and base recognition abilities. To the best of our knowledge, this is the first report in which a fully modified TFO accurately recognizes a complementary DNA duplex having a mixed sequence under neutral conditions.

Visible room-temperature phosphorescence of pure organic crystals via a radical-ion-pair mechanism.

The afterglow of phosphorescent compounds can be distinguished from background fluorescence and scattered light by a time-resolved observation, which is a beneficial property for bioimaging. Phosphorescence emission accompanies spin-forbidden transitions from an excited singlet state through an excited triplet state to a ground singlet state. Since these intersystem crossings are facilitated usually by the heavy-atom effect, metal-free organic solids are seldom phosphorescent, although these solids have recently been refurbished as low-cost, eco-friendly phosphorescent materials. Here, we show that crystalline isophthalic acid exhibits room-temperature phosphorescence with an afterglow that lasts several seconds through a nuclear spin magnetism-assisted spin exchange of a radical ion pair. The obvious afterglow that facilitates a time-resolved detection and the unusual phosphorescence mechanism that enables emission intensification by nuclear spin managements are promising for exploiting the phosphorescence materials in novel applications such as bioimaging.

The Effect of Coatings on the Affinity of Lanthanide Nanoparticles to MKN45 and HeLa Cancer Cells and Improvement in Photodynamic Therapy Efficiency.

An improvement in photodynamic therapy (PDT) efficiency against a human gastric cancer cell line (MKN45) with 5-aminolevulinic acid (ALA) and lanthanide nanoparticles (LNPs) is described. An endogenous photosensitizer, protoporphyrin IX, biosynthesized from ALA and selectively accumulated in cancer cells, is sensitizable by the visible lights emitted from up-conversion LNPs, which can be excited by a near-infrared light. Ten kinds of surface modifications were performed on LNPs, NaYF4(Sc/Yb/Er) and NaYF4(Yb/Tm), in an aim to distribute these irradiation light sources near cancer cells. Among these LNPs, only the amino-functionalized LNPs showed affinity to MKN45 and HeLa cancer cells. A PDT assay with MKN45 demonstrated that amino-modified NaYF4(Sc/Yb/Er) gave rise to a dramatically enhanced PDT effect, reaching almost perfect lethality, whereas NaYF4(Yb/Tm)-based systems caused little improvement in PDT efficiency. The improvement of PDT effect with the amino-modified NaYF4(Sc/Yb/Er) is promising for a practical PDT against deep cancer cells that are reachable only by near-infrared lights.

A monogalactosyldiacylglycerol synthase found in the green sulfur bacterium Chlorobaculum tepidum reveals important roles for galactolipids in photosynthesis.

Monogalactosyldiacylglycerol (MGDG), which is conserved in almost all photosynthetic organisms, is the most abundant natural polar lipid on Earth. In plants, MGDG is highly accumulated in the chloroplast membranes and is an important bulk constituent of thylakoid membranes. However, precise functions of MGDG in photosynthesis have not been well understood. Here, we report a novel MGDG synthase from the green sulfur bacterium Chlorobaculum tepidum. This enzyme, MgdA, catalyzes MGDG synthesis using UDP-Gal as a substrate. The gene encoding MgdA was essential for this bacterium; only heterozygous mgdA mutants could be isolated. An mgdA knockdown mutation affected in vivo assembly of bacteriochlorophyll c aggregates, suggesting the involvement of MGDG in the construction of the light-harvesting complex called chlorosome. These results indicate that MGDG biosynthesis has been independently established in each photosynthetic organism to perform photosynthesis under different environmental conditions. We complemented an Arabidopsis thaliana MGDG synthase mutant by heterologous expression of MgdA. The complemented plants showed almost normal levels of MGDG, although they also had abnormal morphological phenotypes, including reduced chlorophyll content, no apical dominance in shoot growth, atypical flower development, and infertility. These observations provide new insights regarding the importance of regulated MGDG synthesis in the physiology of higher plants.

Sugar-attached upconversion lanthanide nanoparticles: a novel tool for high-throughput lectin assay.

To create a novel high-throughput lectin assay (HTPLA) method based on the emission of a luminophore by highly penetrable near-infrared excitation, sugar-attached upconversion lanthanide nanoparticles (LNPs) were synthesized as a tool to highlight the aggregates caused by the sugar-mediated specific bridging between LNP and lectin. The emissions from a mannose-coated LNP in the aggregates with a mannose-binding lectin were much stronger than those from the non-aggregated samples, being sensitive enough for HTPLA. A galactose-coated LNP was also applicable to a macrophage aggregation assay for the sugar specificity of its surface lectin.

Mapping the diffusion pattern of 1O2 along DNA duplex by guanine photooxidation with an appended biphenyl photosensitizer.

To realize nucleic acid-targeting photodynamic therapy, a photosensitizer should be attached at the optimal position on a complementary oligonucleotide, where a guanine photooxidation is maximized. Here we show the photooxidation of 22 DNA duplexes with varied lengths between a 1O2-generating biphenyl photosensitizer attached at a midchain thymine in a strand and the single guanine reactant in the other strand. The best photooxidation efficiencies are achieved at 9, 10, and 21 base intervals, which coincides with the pitch of 10.5 base pairs per turn in a DNA duplex. The low efficiencies for near and far guanines are due to quenching of the biphenyl by guanine and dilution of 1O2 by diffusion, respectively. The 1O2-diffusion mapping along DNA duplex provides clues to the development of efficient and selective photosensitizer agents for nucleic acid-targeting photodynamic therapy, as well as an experimental demonstration of diffusion of a particle along cylindrical surface in molecular level.

Mangostin enhances efficacy of aminolevulinic acid-photodynamic therapy against cancer through inhibition of ABCG2 activity.

BACKGROUND: Aminolevulinic acid-photodynamic therapy (ALA-PDT) is gaining attention as a potential method for treating select cancers due to its high specificity and low side effect feature. ALA enters cancer cells and accumulate as protoporphyrin IX (PpIX), which will then trigger phototoxicity following light irradiation. However, it is reported that some cancer cells have reduced efficacy of ALA-PDT due to high expression of ABCG2, a transporter involved in the PpIX efflux. In this study, we evaluated the effect of mangostin, a natural compound containing anti-tumor property, on the efficacy of ALA-PDT against cancer and the mechanism involved. METHODS: We utilized TMK1 gastric cancer cell line, which has high ABCG2 expression, to evaluate the PpIX accumulation and phototoxicity exerted by ALA and mangostin co-addition. RESULTS: We found that co-addition of ALA and mangostin significantly increase the phototoxicity and PpIX accumulation in TMK1 cells. We also investigated the effect of mangostin on porphyrin-heme pathway enzymes and ABCG2 and found that the addition of mangostin reduce the activity of ABCG2, reducing PpIX efflux. CONCLUSION: These findings suggest that mangostin enhances the efficacy of ALA-PDT in cancer through inhibition of ABCG2 activity.

Gold nanoparticles conjugated with monosaccharide-modified peptide for lectin detection.

Gold nanoparticles (GNPs) conjugated with monosaccharide-modified peptides have been developed as optical probes for lectin detection. Mannose-modified peptides were designed and conjugated with GNPs. The GNPs with mannose-modified peptide showed remarkable red shift of absorption maximum due to the aggregation with concanavalin A (ConA), a mannose-binding lectin. The aggregation activity of glycopeptide-modified GNPs with ConA depended on the amino acid sequence around the mannose unit of glycopeptides.

2-Oxabutane as a substitute for internal monomer units of oligosaccharides to create lectin ligands.

The synthesis of bioactive oligosaccharides is too tedious to scale up for commercialization. However, structurally simplified glycomimetics are commercializable, if they can be synthesized much more easily than the oligosaccharides while having a comparable bioactivity. In this study, we propose a 2-oxabutane (OB) structure as an imitation of the internal monosaccharide units in oligosaccharides. Two trimannoside and three pentamannoside OB-glycomimics were synthesized in remarkably short steps. Among them, Manα1-OB-2Man 10, a trimannoside mimic, showed eight-fold affinity toward concanavalin A (ConA) relative to methyl mannoside in latex agglutination lectin assay and equilibrium dialysis assay (EDA), while the other mimics showed three- to four-fold affinities. EDA indicated that the bindings between each mimic molecule and a ConA subsite were all in one-to-one stoichiometry and thus these mimics were monovalent ligands, excluding multivalence effect for the high affinities. The strong affinity of 10 could be explained by the occupation of two mannose binding sites of a ConA subsite by its two mannose units. Mimic 10 proved to be even a better ligand for ConA than the natural disaccharide Manα1,2Man, while been much more easy to synthesize, thereby illustrating the potential of the approach here presented.

Large Timescale Interrogation of Neuronal Function by Fiberless Optogenetics Using Lanthanide Micro-particles.

Optogenetics requires implantation of light-delivering optical fibers, as current light-sensitive opsins are activated by visible light, which cannot effectively penetrate biological tissues. Insertion of optical fibers and subsequent photostimulation inherently damages brain tissue, and fiber tethering can restrict animal behavior. To overcome these technical limitations, we developed minimally invasive "fiberless" optogenetics using lanthanide micro-particles (LMPs), which emit up-conversion luminescence in the visible spectrum in response to irradiation with tissue-penetrating near-infrared light. Depolarizing (C1V1) and hyperpolarizing (ACR1) opsins were strongly activated by up-conversion luminescence from green-emitting LMPs both in vitro and in vivo. Using this technique, we successfully manipulated locomotive behavior of mice by activating and inhibiting neurons in the dorsal striatum, at a depth of 2 mm from the brain surface. LMPs were retained and remained functional for >8 weeks at the injection site. Fiberless optogenetics offers opportunities to control neuronal function over longer time frames using freely behaving animals.

A Twist-Assisted Biphenyl Photosensitizer Passable Through Glucose Channel.

While the development of low-molecular-weight drugs is saturating, agents for photodynamic therapies (PDTs) may become alternative seeds in pharmaceutical industry. Among them, orally administrative, cancer-selective, and side effect-free photosensitizers (PSs) that can be activated by tissue-penetrative near-infrared (NIR) lights are strongly demanded. We discovered such a PS from scratch by focusing on a twist-assisted spin-orbit charge transfer intersystem crossing (ISC) mechanism in a biphenyl derivative, which was demonstrated by thorough photophysical studies. The unique ISC mechanism enables the PS to be small and slim so as to pass through glucose transporters and exert a PDT effect selectively on a cancer cell line. The smallness will allow for oral administration and fast clearance, which have been agenda of approved PSs with larger molecular weights. We also demonstrated that our PS was able to be activated with an NIR pulse laser through two-photon excitation.