Kaempferia galanga L. extract and its main component, ethyl p-methoxycinnamate, inhibit the proliferation of Ehrlich ascites tumor cells by suppressing TFAM expression.

Kaempferia galanga L. shows anti-cancer effects; however, the underling mechanism remains unclear. In this study, we explored the underlying mechanism of the anti-cancer effects of Kaempferia galanga L. Kaempferia galanga L. rhizome extracts (KGEs) suppressed Ehrlich ascites tumor cell (EATC) proliferation by inhibiting S-phase progression. The main component of KGE is ethyl p-methoxycinnamate (EMC), which exhibits the same anti-proliferative effect as KGE. Furthermore, EMC induced the downregulation of cyclin D1 and upregulation of p21. EMC also decreased the expression of mitochondrial transcription factor A (TFAM) but did not significantly change mitochondrial DNA copy number and membrane potential. Phosphorylation at Ser62 of c-Myc, a transcription factor of TFAM, was decreased by EMC treatment, which might be due to the suppression of H-ras expression. These results indicate that EMC is the active compound responsible for the anti-cancer effect of KGE and suppresses EATC proliferation by regulating the protein expression of cyclin D1 and p21; TFAM may also regulate the expression of these genes. In addition, we investigated the anticancer effects of KGE and EMC in vivo using EATC bearing mice. The volume of ascites fluid was significantly increased by intraperitoneal administration of EATC. However, the increase in the volume of ascites fluid was suppressed by oral administration of EMC and KGE. This study provides novel insights into the association between the anti-cancer effects of natural compounds and TFAM, indicating that TFAM might be a potential therapeutic target.

Squaraine dye/Ti3C2Tx MXene organic-inorganic hybrids for photocatalytic hydrogen evolution.

MXenes, a new family of 2D nanostructured materials, have been widely studied in the field of artificial photosynthesis due to their outstanding physicochemical properties. In this work, a series of 2,4-bis[4-(N,N-dibutylamino)phenyl] squaraine (SQ) derivatives with different number of hydroxyl groups were hybridized with Ti3C2Tx MXene nanosheets, and the organic-inorganic hybrid photocatalysts were applied for water-splitting hydrogen evolution. The mass ratios of SQ@Ti3C2Tx were optimized to 4 wt% for each SQ, and the best hydrogen evolution reaction (HER) rate of 28.6 µmol h-1 g-1 was achieved by SQ-3 with four OH groups. The photocatalytic ability of the hybrid comes from the outstanding light harvesting of SQ dye, sufficient active sites of Ti3C2Tx, and efficient separation and transfer of the photogenerated charges via heterojunction between SQ aggregates and Ti3C2Tx. This work firstly demonstrates an example of SQ sensitizer combined with MXene for hydrogen generation, which provides a new insight to further explore the MXene-based hybrid nanomaterials for water splitting hydrogen evolution.

Wavelength-Dependent Optical Response of Single Photosynthetic Antenna Complexes from Siphonous Green Alga Codium fragile.

The siphonaxanthin-siphonein-Chl-a/b-protein (SCP) complex from the siphonous green alga Codium fragile is the major light-harvesting complex (LHC) of these alga and is highly homologous to that of green plants (trimeric pigment-protein complex, LHCII). Interestingly, we find remarkable differences in the spectral response from individual SCP complexes when excited at 561 and 639 nm. While excitation in the green spectral range reproduces the common LHCII-like emission features for most of the complexes, excitation in the red spectral range yields a red-shifted emission and a significant reduction of the fluorescence decay time. We hypothesize that the difference in spectral response of SCP to light in the green and red spectral ranges can be associated with the adaption of the algae to their natural habitat under water, where sudden intensity changes are diminished, and excess light features a red-enhanced spectrum that comes at tidal timings.

Preprocess dependence of optical properties of ensembles and single siphonaxanthin-containing major antenna from the marine green alga Codium fragile.

The siphonaxanthin-siphonein-Chl-a/b-protein (SCP) is the light-harvesting complex of the marine alga Codium fragile. Its structure resembles that of the major light-harvesting complexes of higher plants, LHC II, yet it features a reversed Chl a:Chl b ratio and it accommodates other variants of carotenoids. We have recorded the fluorescence emission spectra and fluorescence lifetimes from ensembles and single SCP complexes for three different scenarios of handling the samples. While the data obtained from ensembles of SCP complexes yield equivalent results, those obtained from single SCP complexes featured significant differences as a function of the sample history. We ascribe this discrepancy to the different excitation intensities that have been used for ensemble and single complex spectroscopy, and conclude that the SCP complexes undergo an aging process during storage. This process is manifested as a lowering of energetic barriers within the protein, enabling thermal activation of conformational changes at room temperature. This in turn leads to the preferential population of a red-shifted state that features a significant decrease of the fluorescence lifetime.

Discovery of a novel siphonaxanthin biosynthetic precursor in Codium fragile that accumulates only by exposure to blue-green light.

Photosynthetic organisms adapt to a variety of light conditions. Codium fragile, a macrosiphonous green alga, binds a unique carbonyl carotenoid, siphonaxanthin, to its major photosynthetic light-harvesting complexes, allowing it to utilize dim blue-green light for photosynthesis. Here, we describe the absolute chemical structure of a novel siphonaxanthin biosynthetic precursor, 19-deoxysiphonaxanthin, that accumulates specifically in the photosynthetic antenna only when cultivated under blue-green light. The action spectra of pigment accumulation suggest that siphonaxanthin biosynthesis is regulated by a specific wavelength profile. The results provide clues to a new acclimation mechanism to withstand hours of intense light at low tide and why siphonous algae have been growing invasively on the world's coasts for more than a century.

Structural insights into blue-green light utilization by marine green algal light harvesting complex II at 2.78 Å.

Light-harvesting complex II (LHCII) present in plants and green algae absorbs solar energy to promote photochemical reactions. A marine green macroalga, Codium fragile, exhibits the unique characteristic of absorbing blue-green light from the sun during photochemical reactions while being underwater owing to the presence of pigment-altered LHCII called siphonaxanthin-chlorophyll a/b-binding protein (SCP). In this study, we determined the structure of SCP at a resolution of 2.78 Å using cryogenic electron microscopy. SCP has a trimeric structure, wherein each monomer containing two lutein and two chlorophyll a molecules in the plant-type LHCII are replaced by siphonaxanthin and its ester and two chlorophyll b molecules, respectively. Siphonaxanthin occupies the binding site in SCP having a polarity in the trimeric inner core, and exhibits a distorted conjugated chain comprising a carbonyl group hydrogen bonded to a cysteine residue of apoprotein. These features suggest that the siphonaxanthin molecule is responsible for the characteristic green absorption of SCP. The replaced chlorophyll b molecules extend the region of the stromal side chlorophyll b cluster, spanning two adjacent monomers.

Production of Carotenoids from Cultivated Seaweed.

Cladosiphon (C.) okamuranus, a brown alga endemic to the Nansei Islands, Japan, has been conventionally ingested as food. Nowadays, it is a major aquatic product of the Okinawa Prefecture with an annual production of around 20,000 tons. The life cycle of C. okamuranus comprises the macroscopic sporophyte (algal body) generation and the microscopic gametophyte generation. The germlings in the latter generation can proliferate when floating in seawater. This floating form has been exploited in techniques involved in the commercial production of C. okamuranus seedlings.Brown algae contain fucoxanthin, a carbonyl carotenoid known to have anticancer, anti-obesity, and antidiabetic effect in addition to the anti-oxidation effect. We found that the fucoxanthin content of cultivated floating form of C. okamuranus discoid germlings becomes up to 50 times that of the mature alga. Since the discoid germlings repeatedly grow like microorganisms, although they are large algae, they are utilized to produce fucoxanthin. We optimize the culture conditions by changing the temperature, light intensity, photoperiod, light wavelength, and nutrient salt conditions for optimal fucoxanthin productivity. The cultivation has been successful to industrial plant scale, culminating in the use of 1 ton of cultivating medium.In brown algal cells, fucoxanthin is primarily found bound to the photosynthetic pigment-protein complexes known as fucoxanthin-chlorophyll protein (FCP). Consequently cultivated floating form of C. okamuranus also shows high content of FCP. Isolation and characterization of pigments bound to the FCP were determined precisely, and ultrafast spectroscopies were applied to elucidate the photosynthetic function of fucoxanthin bound to the pigment-protein complexes. This cultivation method has also been applied to the other edible brown algae. We found that the optimal cultivation conditions as well as the yields of fucoxanthin and FCP highly depend on the species.The floating form cultivation was also applied to a large-sized edible green alga, Codium intricatum, which is uniquely producing a carbonyl carotenoid, siphonaxanthin. This has several anti-disease effects and is also a primal photosynthetic pigment which is found bound to photosynthetic antenna complex usually called siphonaxanthin-chlorophyll protein (SCP). We are working on the improvement of productivity, scale-up of production, and development of cultivation technology of new macro algae.

Pigment structure in the light-harvesting protein of the siphonous green alga Codium fragile.

The siphonaxanthin-siphonein-chlorophyll-a/b-binding protein (SCP), a trimeric light-harvesting complex isolated from photosystem II of the siphonous green alga Codium fragile, binds the carotenoid siphonaxanthin (Sx) and/or its ester siphonein in place of lutein, in addition to chlorophylls a/b and neoxanthin. SCP exhibits a higher content of chlorophyll b (Chl-b) than its counterpart in green plants, light-harvesting complex II (LHCII), increasing the relative absorption of blue-green light for photosynthesis. Using low temperature absorption and resonance Raman spectroscopies, we reveal the presence of two non-equivalent Sx molecules in SCP, and assign their absorption peaks at 501 and 535 nm. The red-absorbing Sx population exhibits a significant distortion that is reminiscent of lutein 2 in trimeric LHCII. Unexpected enhancement of the Raman modes of Chls-b in SCP allows an unequivocal description of seven to nine non-equivalent Chls-b, and six distinct Chl-a populations in this protein.

Metabolomic analysis and mass spectrometry imaging after neonatal stroke and cell therapies in mouse brains.

Ischemic brain injury provokes complex, time-dependent downstream pathways that ultimately lead to cell death. We aimed to demonstrate the levels of a wide range of metabolites in brain lysates and their on-tissue distribution following neonatal stroke and cell therapies. Postnatal day 12 mice underwent middle cerebral artery occlusion (MCAO) and were administered 1 × 105 cells after 48 h. Metabolomic analysis of the injured hemisphere demonstrated that a variety of amino acids were significantly increased and that tricarboxylic acid cycle intermediates and some related amino acids, such as glutamate, were decreased. With the exception of the changes in citric acid, neither mesenchymal stem/stromal cells nor CD34+ cells ameliorated these changes. On-tissue visualization with matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) imaging revealed that the signal intensity of glutamate was significantly decreased in the infarct area, consistent with the metabolomic analysis, while its intensity was significantly increased in the peri-infarct area after MCAO. Although cell therapies did not ameliorate the changes in metabolites in the infarct area, mesenchymal stem cells ameliorated the increased levels of glutamate and carnitine in the peri-infarct area. MALDI-MS imaging showed the location-specific effect of cell therapies even in this subacute setting after MCAO. These methodologies may be useful for further investigation of possible treatments for ischemic brain injury.

Molecular Structures and Functions of Chlorophylls-a Esterified with Geranylgeranyl, Dihydrogeranylgeranyl, and Tetrahydrogeranylgeranyl Groups at the 17-Propionate Residue in a Diatom, Chaetoceros calcitrans.

The 17-propionate ester group of chlorophyll(Chl)-a in some oxygenic phototrophs was investigated using HPLC. Chls-a esterified with partially dehydrogenated forms of a phytyl group were found in fully grown cells of a diatom, Chaetoceros calcitrans: geranylgeranyl (GG), dihydrogeranylgeranyl (DHGG), and tetrahydrogeranylgeranyl (THGG). Chls-a bearing such esterifying groups were reported to be found only in greening processes of higher plants, and thus these Chls-a have been thought to be biosynthetic precursors for phytylated Chl-a. Their molecular structures were unambiguously determined using 1H and 13C NMR spectroscopy and mass spectrometry. In particular, the positions of C═C double bonds in DHGG were identified at C2═C3, C6═C7, and C14═C15, and those in THGG were determined to be at C2═C3 and C14═C15. Notably, the present DHGG was different from the previously determined DHGG of bacteriochlorophyll-a in purple bacteria (C2═C3, C10═C11, and C14═C15). Moreover, thylakoid membranes as well as fucoxanthin-chlorophyll-a/c proteins called FCPs were isolated from the diatom, and their Chl-a compositions were analyzed. Chls-a esterified with GG, DHGG, and THGG were detected by HPLC, indicating that such Chls-a were not merely biosynthetic precursors, but photosynthetically active pigments.

Photoprotection vs. Photoinhibition of Photosystem II in Transplastomic Lettuce (Lactuca sativa) Dominantly Accumulating Astaxanthin.

Transplastomic (chloroplast genome-modified; CGM) lettuce that dominantly accumulates astaxanthin grows similarly to a non-transgenic control with almost no accumulation of naturally occurring photosynthetic carotenoids. In this study, we evaluated the activity and assembly of PSII in CGM lettuce. The maximum quantum yield of PSII in CGM lettuce was <0.6; however, the quantum yield of PSII was comparable with that in control leaves under higher light intensity. CGM lettuce showed a lower ability to induce non-photochemical quenching (NPQ) than the control under various light intensities. The fraction of slowly recovering NPQ in CGM lettuce, which is considered to be photoinhibitory quenching (qI), was less than half that of the control. In fact, 1O2 generation was lower in CGM than in control leaves under high light intensity. CGM lettuce contained less PSII, accumulated mostly as a monomer in thylakoid membranes. The PSII monomers purified from the CGM thylakoids bound echinenone and canthaxanthin in addition to ß-carotene, suggesting that a shortage of ß-carotene and/or the binding of carbonyl carotenoids would interfere with the photophysical function as well as normal assembly of PSII. In contrast, high accumulation of astaxanthin and other carbonyl carotenoids was found within the thylakoid membranes. This finding would be associated with the suppression of photo-oxidative stress in the thylakoid membranes. Our observation suggests the importance of a specific balance between photoprotection and photoinhibition that can support normal photosynthesis in CGM lettuce producing astaxanthin.

Light-dependent conformational change of neoxanthin in a siphonous green alga, Codium intricatum, revealed by Raman spectroscopy.

Siphonous green algae, a type of deep-sea green algae, appear olive drab and utilize blue-green light for photosynthesis. A siphonous green alga, Codium (C.) intricatum, was isolated from Okinawa prefecture in Japan, and a clonal algal culture in filamentous form was established. The major light-harvesting antenna was analogous to the trimeric LHCII found in higher plants, but the C. intricatum complex contained an unusual carbonyl carotenoid siphonaxanthin. Culture conditions were optimized to achieve high siphonaxanthin content in intact lyophilized filamentous bodies. Interestingly, the carotenoid composition was different when cultured under high irradiance: all-trans neoxanthin was accumulated in addition to the normal 9'-cis form in whole cell extract. Resonance Raman spectra of intact filamentous bodies, cultured under high- and low-light conditions, confirmed the accumulation of all-trans neoxanthin under high irradiance conditions. A plausible function of the presence of all-trans neoxanthin will be discussed in relation to the regulation against high light stress.

Characterization of the intramolecular transfer state of marine carotenoid fucoxanthin by femtosecond pump-probe spectroscopy.

Fucoxanthin, containing a carbonyl group in conjugation with its polyene backbone, is a naturally occurring pigment in marine organisms and is essential to the photosynthetic light-harvesting function in brown alga and diatom. Fucoxanthin exhibits optical characteristics attributed to an intramolecular charge transfer (ICT) state that arises in polar environments due to the presence of the carbonyl group. In this study, we report the spectroscopic properties of fucoxanthin in methanol (polar and protic solvent) observed by femtosecond pump-probe measurements in the near-infrared region, where transient absorption associated with the optically allowed S2 (1(1)B u (+) ) state and stimulated emission from the strongly coupled S1/ICT state were observed following one-photon excitation to the S2 state. The results showed that the amplitude of the stimulated emission of the S1/ICT state increased with decreasing excitation energy, demonstrating that the fucoxanthin form associated with the lower energy of the steady-state absorption exhibits stronger ICT character.

Widespread binding of FUS along nascent RNA regulates alternative splicing in the brain.

Fused in sarcoma (FUS) and TAR DNA-binding protein 43 (TDP-43) are RNA-binding proteins pathogenetically linked to amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD), but it is not known if they regulate the same transcripts. We addressed this question using crosslinking and immunoprecipitation (iCLIP) in mouse brain, which showed that FUS binds along the whole length of the nascent RNA with limited sequence specificity to GGU and related motifs. A saw-tooth binding pattern in long genes demonstrated that FUS remains bound to pre-mRNAs until splicing is completed. Analysis of FUS(-/-) brain demonstrated a role for FUS in alternative splicing, with increased crosslinking of FUS in introns around the repressed exons. We did not observe a significant overlap in the RNA binding sites or the exons regulated by FUS and TDP-43. Nevertheless, we found that both proteins regulate genes that function in neuronal development.

Ultrafast excited state dynamics of spirilloxanthin in solution and bound to core antenna complexes: identification of the S∗ and T1 states.

Ultrafast excited state dynamics of spirilloxanthin in solution and bound to the light-harvesting core antenna complexes from Rhodospirillum rubrum S1 were investigated by means of femtosecond pump-probe spectroscopic measurements. The previously proposed S∗ state of spirilloxanthin was clearly observed both in solution and bound to the light-harvesting core antenna complexes, while the lowest triplet excited state appeared only with spirilloxanthin bound to the protein complexes. Ultrafast formation of triplet spirilloxanthin bound to the protein complexes was observed upon excitation of either spirilloxanthin or bacteriochlorophyll-a. The anomalous reaction of the ultrafast triplet formation is discussed in terms of ultrafast energy transfer between spirilloxanthin and bacteriochlorophyll-a.

Femtosecond stimulated Raman spectroscopy of the dark S1 excited state of carotenoid in photosynthetic light harvesting complex.

Vibrational dynamics of the excited state in the light-harvesting complex (LH1) have been investigated by femtosecond stimulated Raman spectroscopy (FSRS). The native and reconstituted LH1 complexes have same dynamics. The ν(1) (C=C stretching) vibrational mode of spirilloxanthin in LH1 shows ultrafast high-frequency shift in the S(1) excited state with a time constant of 0.3 ps. It is assigned to the vibrational relaxation of the S(1) state following the internal conversion from the photoexcited S(2) state.

Polarization angle dependence of stark absorption spectra of spirilloxanthin bound to the reconstituted LH1 complexes using LH1-subunits isolated from the purple photosynthetic bacterium Rhodospirillum rubrum.

Reconstituted LH1 complexes were prepared using the LH1 subunit-type complexes, isolated from the purple photosynthetic bacterium Rhodospirillum (Rs.) rubrum, and purified all-trans spirilloxanthin. Stark absorption spectra of spirilloxanthin bound to both the native and reconstituted LH1 complexes were compared in different polarization angles (χ) against the external electric field. From the polarization angle dependence of the Stark absorption spectra, two angles were determined in reference to the direction of transition dipole moment (m) of spirilloxanthin: one is the change in polarizability upon photoexcitation (Δα), θ(Δα) and the other is the change in static dipole moment upon photoexcitation (Δµ), θ(Δµ). Despite the symmetric molecular structure of all-trans spirilloxanthin, its Stark absorption spectra show pronounced values of Δµ. This large Δµ values essentially caused by the effect of induced dipole moment through Δα both in the cases for native and reconstituted LH1 complexes. However, slightly different values of θ(Δα) and θ(Δµ) observed for the native LH1 complex suggest that spirilloxanthin is asymmetrically distorted when bound to the native LH1 complex and gives rise to intrinsic Δµ value.

Isolation and purification of the major photosynthetic antenna, fucoxanthin-Chl a/c protein, from cultured discoid germilings of the brown Alga, Cladosiphon okamuranus TOKIDA (Okinawa Mozuku).

A chlorophyll c binding membrane intrinsic light-harvesting complex, the fucoxanthin-chlorophyll a/c protein (FCP), was isolated from cultured discoid germilings of an edible Japanese brown alga, Cladosiphon (C.) okamuranus TOKIDA (Okinawa Mozuku in Japanese). The discoid germiling is an ideal source of brown algal photosynthetic pigment-protein complexes in terms of its size and easiness of cultivation on a large scale. Ion-exchange chromatography was crucial for the purification of FCP from solubilized thylakoid proteins. The molecular weight of the purified FCP assembly was estimated to be ~56 kDa using blue native-PAGE. Further subunit analyses using 2D-PAGE revealed that the FCP assembled as a trimer consisting of two distinguishable subunits having molecular weights of 18.2 (H) and 17.5 (L) kDa. Fluorescence and fluorescence-excitation spectra confirmed that the purified FCP assembly was functionally intact.

New insights into the structure of the reaction centre from Blastochloris viridis: evolution in the laboratory.

Newly determined crystal structures of the photosynthetic RC (reaction centre) from two substrains of the non-sulfur purple bacterium Blastochloris viridis strain DSM 133, together with analysis of their gene sequences, has revealed intraspecies evolutionary changes over a period of 14 years. Over 100 point mutations were identified between these two substrains in the four genes encoding the protein subunits of the RC, of which approximately one-fifth resulted in a total of 16 amino acid changes. The most interesting difference was in the M subunit where the change from a leucine residue to glycine in the carotenoid-binding pocket allowed NS5 (1,2-dihydroneurosporene) to adopt a more sterically favoured conformation, similar to the carotenoid conformation found in other related RCs. The results of the present study, together with a high rate of mutations in laboratory bacterial cultures described recently, suggest that bacteria evolve faster than has been generally recognized. The possibility that amino acid changes occur within protein sequences, without exhibiting any immediately observable phenotype, should be taken into account in studies that involve long-term continuous growth of pure bacterial cultures. The Blc. viridis RC is often studied with sophisticated biophysical techniques and changes such as those described here may well affect their outcome. In other words, there is a danger that laboratory-to-laboratory variation could well be due to different groups not realising that they are actually working with slightly different proteins. A way around this problem is suggested.