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The cytological characteristics of major green-tide-forming green algae Ulva prolifera collected from Yellow Sea were studied through cutting segments, long time low temperature or dark treatments. After being dried in the shade and preserved at -20°C for 30 days, the U. prolifera was cultured at 4°C in sterilized seawater under 40 μmol photons m-2·s-1 light intensity for 120 days, results indicated that the plastid of U. prolifera continuously shrank with the extension of treatment, and most cells turned white and died, only a small amount of cells still contained a few of visible inclusions at the 120d of treatment. Then those samples were transferred to 20°C and 40 μmol photons m-2·s-1 condition for recovery cultivation, after about 10 days, some recovery cells were observed in the thallus, and those cells developed to young thallus gradually and released germ cells almost in the same time. After about 60 days of recovery cultivation, the newly-grown green thallus broke through the original dead thallus, and the germ cells also grew to new individual thallus. Before dark treatment, the U. prolifera cells were filled with plastid, contained visible starch grain and discernible cell outlines, while after 120 days of dark treatment, the plastid shrank and degraded together with the disappearance of cell inclusions, and the cell outlines also blurred, then those samples were transferred to optimal culture conditions at 20°C in 40 μmol photons m-2·s-1 light intensity, and 15 days later, newly-grown cells appeared on the almost dead thallus, these cells divided continuously and grew to young thallus, and those newly-grown thallus also generated active germ cells, which developed to new thallus that cytologically identical to the original thallus. Observation of chopped tissue of U. prolifera cultivated at 20°C, 40 μmol m-2·s-1 showed that the morphological upper part cells turned to germ cells first, those germ cells including gametophyte and sporophyte, which released later and grew to new individual thallus. These findings provided cytological evidences for how U. prolifera live through stress conditions such as low temperature, darkness, and also useful for understanding the mechanism of the occurrence of green tide.

Conchocelis cultivation is a crucial step in the industrial growth of Pyropia (laver). However, our knowledge of the seawater microbiology community in Pyropia seedling pools is limited. This study investigated the temporal dynamics in seedling pool microbiota before and after outbreaks of yellow spot disease in Pyropia yezoensis conchocelis. A total of 18 water samples were collected to assess seedling pool health status, followed by the sequencing of microbiota 16S ribosomal RNA (rRNA). Analysis of 16,760,224 16S rRNA gene sequences indicated that the core microbe group comprised 207 genera. The microbial diversity of samples gradually increased from May to July. The bacterial diversity of seawater samples from diseased P. yezoensis seedling pools was significantly higher than samples from healthy seedling pools. It is noteworthy that each pool group had a distinctive microbial community composition. The relative abundance of two dominant bacteria genera, Sediminicola and Roseivirga, increased over time in healthy seedling pools, peaking in August, while Vibrio remained at constantly low levels. However, the abundance of Vibrio and Polaribacter increased sharply in diseased P. yezoensis seedling pools and decreased after fresh seawater exchange. This indicates that these bacteria are probably associated with the filament disease. Many disease-associated bacteria are negatively related to the HCO3 − concentration in seawater and positively related to pH. Changes in bacteria and environmental factors reflect the characteristics of microbial community succession in seedling pools and therefore have potential to be used as biomarkers and environmental signals for Pyropia disease monitoring.

Summary The role of SRY-related high-mobility-group box (SOX) 12 in leukaemia progression and haematopoiesis remains elusive. This study aimed to examine the expression and function of SOX12 in acute myeloid leukaemia (AML) using human myeloid leukaemia samples and the acute myeloid cell line THP1. Mononuclear cells were isolated from the bone marrow of AML patients and healthy donors. SOX12 expression in haematopoietic cells was evaluated by reverse transcription polymerase chain reaction (RT-PCR). SOX12 short hairpin RNAs (shRNAs) were transduced into THP1 cells, and gene knockdown was confirmed by quantitative RT-PCR and Western blot analysis. SOX12 was preferentially expressed in CD34+ cells in AML patients. The THP1 cells transduced with SOX12 shRNAs exhibited significantly reduced SOX12 expression and cell proliferation. SOX12 knockdown had no effect on apoptosis, but it induced cell cycle arrest at G1 phase and reduced the number of colonies. The transduced THP1 and primary AML cells were reconstituted in non-obese diabetic-severe combined immunodeficient (NOD/SCID) mice, and their numbers were significantly reduced 6–12 weeks after transplantation. The mRNA and protein levels of β-catenin were significantly diminished following SOX12 knockdown, accompanied by a decrease in TCF/Wnt activity. SOX12 may be involved in leukaemia progression by regulating the expression of β-catenin and then interfering with TCF/Wnt pathway, which may be a target for AML.

High expression of the oncogene ecotropic viral integration site-1 (EVI-1) is an independent negative prognostic indicator of survival in leukemia patients. This study aimed to examine the effects of arsenic trioxide (ATO) on EVI-1 in acute myeloid leukemia (AML). Mononuclear cells were isolated from the bone marrow and peripheral blood of AML patients and healthy donors. EVI-1 expression in hematopoietic cells was evaluated by RT-qPCR and Western blot analysis. EVI-1 was highly expressed in both primary AML and leukemia cell lines (THP-1 and K562). ATO down-regulated EVI-1 mRNA in zebrafish in vivo as well as in primary leukemia cells and THP-1 and K562 cells in vitro. Additionally, ATO treatment induced apoptosis, down-regulated both EVI-1 mRNA and oncoprotein expression, increased the expression of pro-apoptosis proteins, and decreased the expression of anti-apoptotic proteins in leukemia cells in vitro. EVI-1 expression in leukemia cells (THP-1 and K562) transduced with EVI-1 siRNA was significantly reduced. Silencing EVI-1 had a significant effect on the activation of the JNK pathway and the induction of leukemia cell apoptosis. ATO may downregulate EVI-1 mRNA and oncoprotein levels and block the inhibitory effects of EVI-1 on the JNK pathway, which activates the JNK apoptotic pathway, thereby leading to the apoptosis of EVI-1 in AML patients.

Filamentous Bangia, which are distributed extensively throughout the world, have simple and similar morphological characteristics. Scientists can classify these organisms using molecular markers in combination with morphology. We successfully sequenced the complete nuclear ribosomal DNA, approximately 13 kb in length, from a marine Bangia population. We further analyzed the small subunit ribosomal DNA gene (nrSSU) and the internal transcribed spacer (ITS) sequence regions along with nine other marine, and two freshwater Bangia samples from China. Pairwise distances of the nrSSU and 5.8S ribosomal DNA gene sequences show the marine samples grouping together with low divergences (00.003; 0–0.006, respectively) from each other, but high divergences (0.123–0.126; 0.198, respectively) from freshwater samples. An exception is the marine sample collected from Weihai, which shows high divergence from both other marine samples (0.063–0.065; 0.129, respectively) and the freshwater samples (0.097; 0.120, respectively). A maximum likelihood phylogenetic tree based on a combined SSU-ITS dataset with maximum likelihood method shows the samples divided into three clades, with the two marine sample clades containing Bangia spp. from North America, Europe, Asia, and Australia; and one freshwater clade, containing Bangia atropurpurea from North America and China.

The ribosomal intergenic spacer (IGS) variation was compared for the nine main cultivated varieties of Pyropia yezoensis in China obtained from the Chinese National Pyropia Germplasm Repository. The full length of the IGS is 5984 bp including several sequence repeats and some complex structures. The sequence analysis of four partial IGS amplicons ranged from 3628 bp to 3776 bp; and the G+C content is 52.5%. Through similarity analysis and multiple sequence alignment of these partial IGS amplicons from the nine varieties, we have found 278 variable sites which account for approximately 7.5% of the whole IGS length. Both comparison of partial IGS sequences and phylogenetic analysis indicate high levels of genetic variation among different P. yezoensis cultivars in different areas of China. In comparison to the IGS of P. haitanensis, this study highlights the important function of classification of IGS in P. yezoensis. Therefore, the IGS sequence may be used to identify intraspecific variation in P. yezoensis.

The complete nuclear ribosomal DNA (nrDNA) cistron sequence of Pyropia yezoensis has been sequenced, of which each unit is composed of the intergenic spacer (IGS), the small-subunit (SSU) ribosomal RNA (rRNA) gene, the internal transcribed spacer 1 (ITS1), the 5.8S rRNA gene, the internal transcribed spacer 2 (ITS2), and the large-subunit (LSU) rRNA gene. This cistron occurs in tandem repeats that can number in 100s to 1000s. Furthermore, in the analysis of the sequence data, we determined that introns were present in the small-subunit rRNA genes, for which the exon sequences (1834 bp) are almost identical. The intron numbers ranging from zero to two in the small-subunit rRNA genes are different between multiple individuals. However, the full length of the large-subunit rRNA genes is 4770 bp without any introns, and the 5.8S rRNA gene is 159 bp. Moreover, the 5′ and 3′ terminal sequences of the genic regions of the ribosomal cistron are extremely conservative even among various species in red alga. In addition, the range for ITS1 is 371–372 bp and for ITS2 is 532 to 535 bp. The IGS is 5984 bp in length and possesses upstream direct repeats, downstream repeats, and middle inverted repeats that make sequencing untoward. This is the first time that the complete nucleotide of the IGS region of P. yezoensis has been sequenced. The provision of the full length of nrDNA cistron will offer useful information to be widely utilized for identification, taxonomic relationships, and phylogenies in red algae.

The photosynthetic characteristics of a Pyropia haitanensis red mutant mutated from a wild-type strain using chemical mutagens were studied. The aim was to compare the differences in photosynthetic characteristics between the red mutant and the wild type, and then to analyse the possibility of the red mutant as breeding material. The results indicated that the cytoplasm thickness of the red mutant and the wild type were similar, whereas the cell wall of the red mutant was significantly thinner than that of the wild type. Additionally, the chlorophyll a extract absorption spectra of both strains exhibited no obvious difference, whereas the absorption peaks at 498, 545, and 565 nm (which belong to phycoerythrin, PE) in the phycobiliprotein extract absorption spectra of the red mutant were clearly higher than those of the wild type. The PE content per area of the red mutant thallus was significantly higher than that of the wild type, and the ratios of PE to phycocyanin (PC) and PE to chlorophyll a were significantly higher in the red mutant than in the wild type. The net photosynthetic oxygen evolution rates of the red mutant and wild type cultured under different light intensities demonstrated that the oxygen evolution rates of all samples decreased with increasing light intensities, whereas the oxygen evolution rate of the red mutant was higher than that of the wild type under the same light intensities. The light response curves also indicated that the light-saturated photosynthetic rates (P max), light saturation points (LSPs), and light compensation points (LCPs) of both strains decreased to different degrees with increasing light intensities and that the P max and LSP values of the red mutant were significantly higher than those of the wild type under identical light intensities. In contrast, there were no obvious differences in the respiration rate (R d) and in the LCP. The F v/F m values of both strains were similar before light treatment, whereas the F v/F m of the red mutants after culture under different light intensities (120 and 200 μmol photons m−2 s−1) for 3 days were significantly higher than those values of the wild type. Moreover, ∆F/F m′ also exhibited a similar variation. Based on these results, the high content of PE in the P. haitanensis red mutant causes a steady red phenotype, and the increase in the PE concentration and the decrease in the thickness of the thallus may enhance its photosynthetic physiological activity. Thus, the red mutant of P. haitanensis may provide good material for Pyropia breeding.

Carotenoid metabolism in red algae is not well understood. Geranylgeranyl diphosphate (GGPP), synthesized by GGPP synthase (GGPS), is a precursor for the biosynthesis of many biologically important metabolites, including carotenoids and chlorophylls. GGPSs have been functionally characterized in many organisms, but not in species of the primitive red algal order Bangiales. Here, we cloned and characterized the gene encoding GGPS (PuGGPS) in Pyropia umbilicalis (Bangiales). PuGGPS encodes a protein of 345 amino acids with an N-terminal transit peptide. The catalytic activity of PuGGPS for the production of GGPP was verified by a color complementation assay in Escherichia coli and subsequent high-performance liquid chromatography analysis. Homology modeling of PuGGPS showed that its tertiary structure resembles that of other known GGPSs and that this structure allows for the precise docking of the enzymatic product of PuGGPS, GGPP. When leafy thalli of P. umbilicalis were treated with norflurazon, an inhibitor of the key carotenoid metabolism enzyme phytoene desaturase, the expression of PuGGPS increased by twofold compared with that of the control in the first 2 h, suggesting a prompt response to metabolic perturbation. Prolonged norflurazon treatment failed to increase PuGGPS expression. Sequence analysis showed that PuGGPS shares seven conserved motifs with other previously identified GGPSs from different organisms, including two aspartate-rich GGPS signature motifs. Phylogenetic analysis also indicated that PuGGPS is a member of the type II GGPSs found in eubacteria and plants.

A molecular taxonomic study was undertaken for the first time of the bladed Bangiales of the mainland coast of China (Northwest Pacific) based on sequence data of 201 plastid rbcL and 148 nuclear 18S sequences of historical and contemporary specimens. The results revealed that only one genus of bladed Bangiales, Pyropia, was present along Chinese coast. Species delimitation was determined using two empirical methods: the Automatic Barcode Gap Discovery (ABGD) and General Mixed Yule Coalescence (GMYC) coupled with detection of monophyly in tree reconstruction. At least fourteen species of Pyropia were recovered. Six species were confirmed that had been recorded previously based on morphology (Py. suborbiculata, Py. yezoensis, Py. haitanensis, Py. katadae, Py. tenera and Py. acanthophora), three species were recorded from China for the first time (Py. kinositae, Py. pseudolinearis and Py. tanegashimensis), and five cryptic species that did not match any molecular sequences were also discovered. The phylogeny of the concatenated rbcL and 18S dataset resolved three singletons and four clades. Each clades has a strong trend towards occupying a biogeographic region, but they are not confined to them. A transoceanic and antitropical pattern of distribution was found for Pyropia at both the subgeneric and species level. This together with high biodiversity (ca. 30% of all known Pyropia species) indicates that the Northwest Pacific might act as a centre of origin for modern distribution of Pyropia since the early Cenozoic.

The metabolic pathway of 8-heptadecene in red algae was investigated. The results showed that the amounts of 8-heptadecene in the primitive bangiophycidean red algae Pyropia and Bangia were 30–50 % of the volatile compounds, much higher than that in the green alga Ulva pertusa, the brown alga Sargassum thunbergii and the florideophycidean red alga Gracilaria lemaneiformis. Studies on the metabolism of 8-heptadecene in Pyropia found that its enzymatic system has no significant catalytic activity on palmitic acid, oleic acid, linoleic acid, linolenic acid, and arachidonic acid. However, the isolated enzymatic solution showed activity in the presence of eicosapentaenoic acid. This activity produced about four times the amount of 8-heptadecene compared with other substrates and the control, indicating the solution had a specific catalytic function for eicosapentaenoic acid. Furthermore, the enzyme solution was strongly inhibited by NaN3 but not by phenidone and phenanthroline suggesting that the enzyme is structurally related to heme protein. Thus, it is believed that a constant amount of 8-heptadecene is maintained in the primitive red alga Pyropia and the 8-heptadecene is a metabolite of eicosapentaenoic acid and may catalyzed by enzymes including a heme lipoxygenase-like enzyme.

The photosynthetic characteristics of thalli of cultured Pyropia yezoensis strains collected in January, February, and March in seaweed cultivation area of South China Yellow Sea were studied. Results showed that the maximum quantum efficiency (F v/F m) of all P. yezoensis thallus collected at different times was 0.65. The actual quantum efficiency (ΔF/F m′) of samples in January was the lowest of all samples, while the ΔF/F m′ of samples in March was significantly higher than those in January and February. The increase of temperature and photosynthetic pigments ratios of phycoerythrin and chlorophyll a (PE/Chla) and phycocyanin and chlorophyll a (PC/Chla) from January to March may be the important reasons for the increase in light use efficiency of thallus; although the thallus in March was significantly thicker than in January which may have reduced the light energy absorbed by photosynthetic pigments, the increase of relative high energy use efficiency also helped to maintain the photosynthetic oxygen evolution rate in March. The thicker thallus also reduced photodamage, and the thallus area was increased obviously in March, so the growth rate of thallus in March was over 35 % higher than that in February. Our research indicates that the photosynthetic characteristics of P. yezoensis strains thalli have a close relationship with their growth stage and environmental factors especially temperature, and those photosynthetic characteristics are also reflected in the growth rate of the thalli.

In this study, chlorophyll fluorescence parameters (∆F/F m′, F v/F m) and oxygen evolution of female vegetative tissues of Porphyra katadai var. hemiphylla in unisexual culture (FV) and in mixed culture with male vegetative tissues (FV-M) were followed at 5–20 °C, 10 and 80 μmol photons m−2 s−1. The formation of reproductive tissues was closely correlated with decreasing photosynthetic activities. At the same temperature the tissues cultured under 80 μmol photons m−2 s−1 showed a greater extent of maturation than those under 10 μmol photons m−2 s−1, and their decrease in photosynthesis was also larger. Under the same light intensity the extent of maturation increased with increasing temperature, and both cultures showed higher values of ∆F/F m′ and F v/F m at 10 and 15 °C, while their oxygen evolution became negative at 15–20 °C during the later period. Under the same culture condition the maturation of FV-M culture was relatively faster than that of FV culture, while their photosynthetic activity, especially ∆F/F m′, was lower.

Pyropia yezoensis (Ueda) M. S. Hwang et H. G. Choi (previously called Porphyra yezoensis) is an economically important alga. The blades generated from conchospores are genetic chimeras, which are not suitable for genetic similarity analysis. In this study, two types of blades from a single filament of P. yezoensis sporophyte filament were obtained. One type, ConB, consisted of 40 blades that had germinated from conchospores. The other type, ArcB, consisted of 88 blades that had germinated from archeospores released from ConB. Both of them were analyzed by amplified fragment length polymorphism. The low genetic similarity levels for both conchospore-germinated and archeospore-germinated blades demonstrated that the conchcelis we used was cross-fertilized. Furthermore, a higher polymorphic loci ratio (98.6%) was detected in ArcB than in ConB (80.7%), and the average genetic similarity of ArcB (average 0.61) was lower than that of ConB (average 0.71). These differences indicated that genetic analysis using ArcB gives more accurate results.

Photosystem II photochemistry and phycobiliprotein (PBP) genes of red algaeKappaphycus alvarezii, raw material ofκ-carrageenan used in food and pharmaceutical industries, were analyzed in this study. Minimum saturating irradiance (Ik) of this algal species was less than 115 μmol m−2 s−1. Its actual PSII efficiency (yield II) increased when light intensity enhanced and decreased when light intensity reached 200 μmol m−2 s−1. Under dim light, yield II declined at first and then increased on the fourth day. Under high light, yield II retained a stable value. These results indicate thatK. alvareziiis a low-light-adapted species but possesses regulative mechanisms in response to both excessive and deficient light. Based on the PBP gene sequences,K. alvarezii, together with other red algae, assembled faster and showed a closer relationship with LL-Prochlorococcuscompared to HL-Prochlorococcus. Many amino acid loci in PBP sequences ofK. alvareziiwere conserved with those of LL-Prochlorococcus. However, loci conserved with HL-Prochlorococcusbut divergent with LL-Prochlorococcuswere also found. The diversities of PE and PC are proposed to have played some roles during the algal evolution and divergence of light adaption.

Photosynthetic characteristics of four Porphyra yezoensis Ueda [a taxonomic synonym of Pyropia yezoensis (Ueda) M. S. Hwang et H. G. Choi] strains in conchocelis phase were investigated and compared with one wildtype of P. yezoensis and two strains of Porphyra haitanensis T. J. Chang et B. F. Zheng [a taxonomic synonym of Pyropia haitanensis (T. J. Chang et B. F. Zheng) N. Kikuchi et M. Miyata]. Results showed that experimental strains had higher contents of chl a and carotenoids, but a lower content of total phycobiliproteins than the wildtype. Meanwhile, photochemical efficiency of PSII was measured using pulse amplitude modulation (PAM) fluorometry technology. The value of PSII photosynthetic parameters of P. yezoensis strains were all higher than the wild strain, and the maximal quantum yields (Fv /Fm ), effective quantum yields Y(II), and relative photosynthetic electron transport rates (rETR) of P. haitanensis were higher than those of P. yezoensis. The present study verified the possibility of selective breeding of P. yezoensis using the filamentous sporophyte instead of the gametophytic thallus, the advantages being (i) nonrequirement of control of life cycle and (ii) direct and rapid cultivar improvement by artificial selection. We consider the method to be a promising technique for selective breeding of P. yezoensis cultivars.

The levels of photosynthetic pigments and chlorophyll fluorescence of Porphyra yezoensis strains selected from high-light environments were investigated. Sutong and Sulian strains originated from the same high-light environment but were selected from different sites on the Yellow Sea coast of Jiangsu Province, China. In January (a low temperature period), the Sulian strain and the WT (a widely cultivated strain) had higher levels of chlorophyll a, phycoerythrin, phycocyanin, and allophycocyanin, and higher actual photochemical efficiency of PSII (ΔF/F m′) than the Sutong strain. This indicated that Sulian and the WT may have better adaptation to low temperature. In March (an optimal temperature period), Sutong had higher levels of photosynthetic pigments and higher ΔF/F m′ than the WT and Sulian strains. This suggested that Sutong had higher light use efficiency at optimal temperatures and that most energy absorbed by PSII was used for photosynthetic electron transport. The differing areas of origin of these strains may have resulted in these differences in temperature adaptation.

The formation of archeospores is characteristic of Porphyra yezoensis Ueda and is important for Porphyra aquaculture. Recently, it has been regarded as a valuable seed source for propagation of thalli in mariculture. Cell wall composition changes are associated with archeospore formation in P. yezoensis. Here, we report changes of cell walls of P. yezoensis during archeospore formation. The surfaces of vegetative cells that were originally smooth became rougher and more protuberant as archeosporangia were formed. Ultimately, the cell walls of archeosporangia ruptured, and archeospores were released from the torn cell walls that were left at distal margins of thalli. With changes in cell walls, both effective quantum yield and maximal quantum yield of the same regions in thalli gradually increased during the transformation of vegetative cells to archeospores, suggesting that the photosynthetic properties of the same regions in thalli gradually increased. Meanwhile, photosynthetic parameters for different sectors of thalli were determined, which included the proximal vegetative cells, archeosporangia, and newly released archeospores. The changes in photosynthetic properties of different sectors of thalli were in accordance with that of the same regions in thalli at different stages. In addition, the photosynthetic responses of archeosporangia to light showed higher saturating irradiance levels than those of vegetative cells. All these results suggest that archeosporangial cell walls were not degraded prior to release but were ruptured via bulging of the archeospore within the sporangium, and ultimately, archeospores were discharged. The accumulation of carbohydrates during archeospore formation in P. yezoensis might be required for the release of archeospores.

In January and February 2010, heavy sea ice formed along the coast of the Bohai Sea and the northern Yellow Sea, China. Intertidal organisms were subjected to serious freezing stress. In this study, we investigated the freezing tolerance of the upper intertidal economic seaweed Porphyra yezoensis. The maximum photochemical efficiency of PS II (F v/F m) in undehydrated thalli remained high after 24 h at −2°C and that in dehydrated thalli decreased in a proportion to thallial water loss. F v/F m dropped sharply after 24 h at −20°C, regardless of absolute cellular water content (AWC). The F v/F m in frozen thalli recovered rapidly at 0–20°C. A wide range of water loss in the thalli enhanced their tolerance to freezing. F v/F m values in undehydrated thalli dropped sharply after 3 d at −2°C or 10 d at −20°C while those in dehydrated thalli (20–53% AWCs) remained at high levels after 9 d at −2°C or 30 d at −20°C. These results indicate that P. yezoensis has high freezing tolerance by means of dehydration during the ebb tide and rapid recovery of F v/F m from freezing. A strategy of P. yezoensis industry to avoid heavy loss during freezing season is discussed based on these findings.

Two strains, which are the free-living conchocelis of Porphyra yezoensis Ueda and Porphyra haitanensis T. J. Chang et B. F. Zheng, and four “interspecific hybridization” strains of these two species are investigated. The rbcL genes of 11 samples were amplified and sequenced, each of which were about 1,400 bp with a good specificity. The results of pair-wise distance matrix and multi-alignment showed that pair-wise distance were small while homologous index was large between strains Y-2066, Y-k2001, and Y-H001. These two indexes showed the same level as the above between strains H-2001, Y-hyC1, and Y-hyC2; however, the distances were larger between three former strains and three latter strains. Cluster trees showed the same trend. Fertile strains appeared after 2 years of culture of unfertile interspecific hybridization conchocelis and were separated from that based on different colors. Our finding that these fertile strains can bear offspring is important for understanding the interspecies hybridization of Porphyra. Molecular analysis of the fertile strains based on rbcL gene showed maternal inheritance. We inferred that somatic recombination was one of the reasons making interspecific hybridization strains fertile.

Pigment mutants were used as genetic markers to study the early development and morphogenesis of blades in four species of Porphyra. In Porphyra haitanensis, P. yezoensis, and P. oligospermatangia, the first two divisions are transverse during conchospore germination, yielding four cells arranged in a line. These species are representative of linear development pattern in Porphyra. Resulting in blades with color sectors vertically arranged. In P. katadai var. hemiphylla, the first division is transverse and the upper cell divides vertically forming two side-by-side cells, and its blades are derived mostly from the upper cell showing a bilateral development pattern with two lateral parts of different colors. In this type of germination, most or the entire blade is derived from the upper cells. Some fronds of P. katadai var. hemiphylla developed in linear pattern. In addition, 9.3% of the conchospore germlings of linear development were produced at 10°C, 15.3% at 15°C, and 38.0% at 20°C for conchospore germlings of P. katadai var. hemiphylla. More linear development occurred at higher temperatures. The results revealed general trends of early developmental patterns and morphogenesis of blades within the genus of Porphyra. Developmental patterns and morphogenesis of blades are under the influence of temperatures.

Bangia fuscopurpurea (Rhodophyta) was cultivated in Putian (Fujian province, China). The characteristics of the life history concerned with cultivation were investigated and the cultivation procedure was presented. The gametophytic phase (thallus) and the sporophytic phase (conchocelis) occurred alternately in the life history of B. fuscopurpurea. Young thalli produced archeospores, and the number depended on the environmental factors. Temperature affected the number of archeospore release and percent of germination, and photo flux density (PFD) mainly affected the time of spore release and germination. Thalli matured from December to February and developed into the conchocelis phase through sexual reproduction. The conchocelis grown in shells had three developmental stages: vegetative conchocelis, conchosporangiall formation and conchospore formation. Pit-connections were present in the first 2 stages but absent after conchospore formation. Vegetative conchocelis and conchosporangial. branches can transform into each other. However, conchospores only developed into the gametophytic phase. Cultivation of B. fuscopurpurea was based on characterization of the life history, consisting of 3 steps: zygotospores collection, indoor cultivation of conchocelis and outdoor cultivation of thalli. Young thalli that developed from conchospores produced numerous archeospores before December. Over 90% of the crop was from the development of archeospores. The results indicated that conchosporelings were a good source of archeospores, and the development of the large quantity of archeospores acted as a more prevailing means to increase the population size. (C) 2008 Elsevier B.V. All rights reserved.

The color mutations in Bangiaceae were investigated by treating the blades, conchocelis and conchospores phase of Bangia sp., Porphyra yezoensis, and P. haitanensis sampled in China with mutagen N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). A high percentage of mutation in different expression characteristics in all three phases were shown within optimum mutagen concentrations. Among mutagenized blades, mutations occurred on single cells, which is a direct outcome of mutation of haploid cells. The mutation of mutagenized conchocelis resulted in a two-step process: low-level expression in conchocelis phase, and high-level expression in progeny, explaining that mutation took place in diploid cells. The mutations of conchospores were expressed immediately at germination of spores, indicating a change in ploidy. This paper reports the process of meiosis and its effect on frond development, and the relation between color mutations and morphological characteristics expressed by mutations in Bangiaceae.

Nuclear divisions of carpospores, conchocelis and conchospores of Porphyra yezoensis, P. haitanensis, P. katadai var. hemiphylla and P. oligospermatangia from China were investigated. The observations showed diploid chromosome numbers of 2n = 6 for P. yezoensis and P. oligospermatangia, and 2n = 10 for P. haitanensis and P. katadai var. hemiphylla. For all four species, somatic pairing of chromosome sets was observed in late prophase. Sister chromosomes separated at anaphase as mitosis took place in carpospores, conchocelis filamentous cells, conchosporangial branch cells and sporangial cells (conchospore formation). Chromosome configurations of tetrad and ring-shaped in conchospore germination were observed, demonstrating the occurrence of meiosis. The characteristics of diploid nuclear division in 2n = 6 species are the same as those of 2n = 10 species. The influence of somatic pairing on nuclear division of diploid cells in Porphyra was discussed.

Background Acute myeloid leukemia (AML) 1-Evi-1 is a chimeric gene generated by the t (3; 21) (q26; q22) translocation, which leads into malignant transformation of hematopoietic stem cells by unclear mechanisms. This in vivo study aimed to establish a stable line of zebrafish expressing the human RUNX1-Evi-1 fusion gene under the control of a heat stress-inducible bidirectional promoter, and investigate its roles in hematopoiesis and hematologic malignancies. Methods We introduced human RUNX1-Evi-1 fusion gene into embryonic zebrafish through a heat-shock promoter to establish Tg(RE:HSE:EGFP) zebrafish. Two males and one female mosaic F0 zebrafish embryos (2.1 %) were identified as stable positive germline transgenic zebrafish. Results The population of immature myeloid cells and hematopoietic blast cells were accumulated in peripheral blood and single cell suspension from kidney of adult Tg(RE:HSE:EGFP) zebrafish. RUNX1-Evi-1 presented an intensive influence on hematopoietic regulatory factors. Consequently, primitive hematopoiesis was enhanced by upregulation of gata2 and scl, while erythropoiesis was downregulated due to the suppression of gata1. Early stage of myelopoiesis was flourishing with the high expression of pu.1, but it was inhibited along with the low expression of mpo. Microarray analysis demonstrated that RUNX1-Evi-1 not only upregulated proteasome, cell cycle, glycolysis/gluconeogenesis, tyrosine metabolism, drug metabolism, and PPAR pathway, but also suppressed transforming growth factor β, Jak-STAT, DNA replication, mismatch repair, p53 pathway, JNK signaling pathway, and nucleotide excision repair. Interestingly, histone deacetylase 4 was significantly up-regulated. Factors in cell proliferation were obviously suppressed after 3-day treatment with histone deacetylase inhibitor, valproic acid. Accordingly, higher proportion of G1 arrest and apoptosis were manifested by the propidium iodide staining. Conclusion RUNX1-Evi-1 may promote proliferation and apoptosis resistance of primitive hematopoietic cell, and inhibit the differentiation of myeloid cells with the synergy of different pathways and factors. VPA may be a promising choice in the molecular targeting therapy of RUNX1-Evi-1-related leukemia.

Background Red algae are primitive photosynthetic eukaryotes, whose spores are ideal subjects for studies of photosynthesis and development. Although the development of red alga spores has received considerable research attention, few studies have focused on the detailed morphological and photosynthetic changes that occur during the early development of tetraspores of Gracilaria vermiculophylla (Ohmi) Papenfuss (Gracilariales, Rhodophyta). Herein, we documented these changes in this species of red algae. Results In the tetraspores, we observed two types of division, cruciate and zonate, and both could develop into multicellular bodies (disks). During the first 84 hours, tetraspores divided several times, but the diameter of the disks changed very little; thereafter, the diameter increased significantly. Scanning electron microscopy observations and analysis of histological sections revealed that the natural shape of the disk remains tapered over time, and the erect frond grows from the central protrusion of the disk. Cultivation of tissue from excised disks demonstrated that the central protrusion of the disk is essential for initiation of the erect frond. Photosynthetic (i.e., PSII) activities were measured using chlorophyll fluorescence analysis. The results indicated that freshly released tetraspores retained limited PSII photosynthetic capabilities; when the tetraspores attached to a substrate, those capabilities increased significantly. In the disk, the PSII activity of both marginal and central cells was similar, although some degree of morphological polarity was present; the PSII photosynthetic capabilities in young germling exhibited an apico-basal gradient. Conclusions Attachment of tetraspores to a substrate significantly enhanced their PSII photosynthetic capabilities, and triggered further development. The central protrusion of the disk is the growth point, may have transfer of nutritive material with the marginal cells. Within the young germling, the hetero-distribution of PSII photosynthetic capabilities might be due to the differences in cell functions.

Reproductive apparatus of Gracilaria/Gracilariopsis lemaneiformis collected from Qingdao city were studied with a light and a transmission electron microscope. The special superficial arrangement of spermatangium for this species was clearly observed, and the ultrastructure of spermatangial development revealed the similar cytodynamic pattern followed by all the Gracilariaceae members developed from spermatangial mother cells to spermatangium. The female reproductive apparatus before fertilization was also observed and trichogyne was found protruding above the cortex, contrary to the earlier reports. Tetrasporangium was formed by an outer cortical cell and the tetraspores became spherical and expended after being released.

We used functional analyses of PNAS-2 by RNAi and RNA overexpression and demonstrated PNAS-2 as an anti-apoptotic gene. PNAS-2 expression was significantly increased in de novo and relapsed acute leukemia patients, but in patients in complete remission, the expression of PNAS-2 decreased to the levels comparable to those found in normal controls. In carcinomas, PNAS-2 expression was not up-regulated, indicating that PNAS-2 overexpression was specific for leukemia. So we hypothesized that PNAS-2 could act as an oncogene and anti-apoptotic gene which probably participates leukemogenesis (Hai-Rong WANG, et al. Oncology, Accepted). To study the subcellular localization of PNAS protein, we constructed PNAS-2-GFP expression plasmids and transfected them into U937 leukemic cell line. We found that PNAS-2-GFP fusion protein not only located in vesicles near the nucleus but also distributed throughout the cytosol instead of Diane’s report that CHMP5/PNAS-2 was found in vesicles near the nucleus in Cos-7 cells. This phenomenon indicates the abnormal location of PNAS-2 protein either in U937 leukemia cells or in Cos-7 cells. For the protein classification and secondary structure prediction system: SOSUI showed that PNAS-2 wasn’t a membranal protein and the function of CHMP5/PNAS-2 protein was involved in the lysosome and MVB (multivesicular body), so PNAS-2 should be a protein in the interior of the MVB and lysosome, thus abnormal location of PNAS-2 protein might occur in U937 leukemic cells. From our data and related articles, PNAS-2 served as an anti-apoptotic gene and located in MVB and lysosome. We suggested that PNAS-2 might execute its anti-apoptotic function via following ways: it might be a protease which could degrade apoptogenic factors such as CatD, GraB and perforin in MVB and lysosome; or it could return CatD, GraB and perforin to the lysosome; or it could stabilize the membrane of MVB and lysosome. When PNAS-2 was set free into the cytosol from MVB structure in leukemic cells, it would enhance its anti-apoptotic function via enhanced above-mentioned ways. Figure Figure Now compelling evidences showed that insufficient apoptosis could result in tumor genesis, thus we thought abnormal subcellular location of PNAS-2 in leukemic cells might participate in leukemogenesis. Antibody microarray was used to study the protein changes after inhibition of PNAS-2, we found cancer suppressor proteins such as p53, Rb increased after PNAS-2 was suppressed, while the rest cancer suppressor proteins did not change, indicating the overexpression of PNAS-2 might participate in tumorigeness by suppressing tumor suppressor proteins. Of notice, Granzyme B-perforin apoptotic pathway was inhibited when PNAS-2 was up-regulated. Granzyme-mediated apoptosis was part of the immune surveillance mechanism where malignantly transformed cells were targeted by CTL and NK cells before cancer could ensue. In conclusion, the abnormal location of PNAS-2 in leukemic cells could enhance its anti-apoptotic function. PNAS-2 might participate in leukemogenesis via its inhibition of apoptosis, cancer suppressor proteins and Granzyme B-perforin pathway.

We recently demonstrated PNAS-2 serving as both an anti-apoptotic gene and an oncogene which is specifically up-regulated in acute leukemic patients (Hai-Rong WANG, et al. Oncology, Accepted). However, its role in apoptotic pathway remains elusive. Antibody microarray was applied to study the apoptosis-associated proteins' changes to elucidate its role in apoptotic pathway. After PNAS-2 was inhibited by RNA interference, caspase3 was up-regulated confirming that PNAS-2 had an anti-apoptotic function. The activation of extrinsic, receptor-mediated apoptotic pathway was characterized by binding a death ligand to the corresponding death receptor. In this study, there were no changes in TNFα, CD95/Fas, TNFR, FADD, TRADD and caspase8 though Fas ligand decreased, while the apoptotic inhibitors such as DcR1 and FLIP were up-regulated, indicating there was no activation in the extrinsic apoptotic pathway. As to the apoptogenic proteins of classical intrinsic apoptotic pathway, there were no changes in Bax and caspase 9 though Bim was down-regulated, while the apoptotic inhibitors such as Bcl-2, Bcl-XL were up-regulated, indicating there was no activation in the intrinsic apoptotic pathway as well. Apart from the two classical apoptotic pathways (intrinsic and extrinsic), Granzyme B (GraB)-perforin apoptotic pathway is one well defined extrinsic accessory way of triggering apoptosis. In our data, GraB and Perforin were up-regulated, suggesting the GraB-Perforin apoptotic pathway was also activated. The apoptosis-induced factor (AIF) and Cathepsin D (CatD) were also up-regulated. AIF could induce apoptosis via a caspase-independent pathway, and Cat D could selectively escape AIF from mitochondria when translocated into the cytosol from lysosome, while other apoptogenic proteins resided in the intermembrane space of mitochondria such as cytochrome c and endonuclease G remained in the mitochondria (Nicolas Bidere, et al. JBC, 2003). Our data indicated that the AIF-induced-caspase-independent pathway was activated when PNAS-2 was inhibited. The changes in protein levels of CatD, AIF and GraB were confirmed by Western Blot. Mannose-6-Phosphate receptor (M6Pr) is the target receptor for GraB and CatD, mediating uptake of the proteases to form vesicles via endocytosis. This receptor also serves to transport newly synthesized GraB and CatD into the vesicles. The vesicles would form a structure named multivesicular body (MVB), which served to sort vesicles' content destined for degradation or routing to the lysosome. CHMP5 (the alias of PNAS-2) located in MVB and lysosome, and the loss of CHMP5/PNAS-2 could inhibit lysosomal degradation (Jae-Hyuck Shim, et al. JCB, 2006). As GraB and CatD could trigger apoptosis when they were effluxed from the lysosome, we hypothesize that the overexpression of PNAS-2 in leukemic patients might execute its anti-apoptotic function by increasing the lysosomal degradation of apoptogenic factors in lysosome such as CatD and GraB. Download : Download high-res image (232KB) Download : Download full-size image Figure