The desoxyribonucleic acid content of animal cells and its evolutionary significance

1. Evidence is summarized for the constancy of DNA content for each set of chromosomes in the various cells of an organism. 2. The DNA contents of the egg and sperm nuclei are the same. 3. A brief survey is given of DNA contents per cell in invertebrates and vertebrates. (a) In invertebrates there is some slight evidence that when primitive and higher forms are compared the amount of DNA per cell is increased in the latter. (b) In fishes there is a tendency for the amount of DNA per cell to remain constant within the different species of a family. (c) The values of DNA per cell in lung fishes, amphibians, reptiles, and birds suggest that in the evolution of these vertebrates there has been a decline in DNA content per cell. 4. Concerning the significance of quantity of DNA per cell in vertebrates: (a) It appears not to be in proportion to the number of different genes in a cell. (b) It may be related to the number of strands in the chromosomes. (c) In homologous cells of different animals it is directly related to the mass of the cell.     

Establishment and characterization of the Bombyx mandarina cell line

A new cell line, designated as NIAS-Boma-529b, was established from the larval fat bodies of Bombyx mandarina (B. mandarina), which is believed to be an ancestor of Bombyx mori (B. mori). This cell line has been cultured for approximately 150 passages during 2 years in an IPL-41 medium supplemented with 10% fetal bovine serum at a constant temperature of 26 °C. The morphology of this line includes adhesive round and spindle-shaped cells. Random-amplified polymorphic DNA analysis (RAPD) using 7 primers and a statistical analysis based on Nei’s genetic distance revealed that this cell line was closely related to B. mori-derived cell lines. An infection study also revealed that this cell line was susceptible to B. mori nucleopolyhedrovirus (BmNPV); however, it had no apparent susceptibility to Autographa californica NPV (AcNPV), which is closely related to BmNPV. Nevertheless, cells infected with AcNPV showed an extensive cytopathic effect (CPE), including a rough cell surface, rounding, nuclear expansion, and cell blebbing. These results suggest that this cell line can be useful to clarify the mechanism of host range determination of BmNPV and AcNPV.     

Cellular interaction between cytotoxic and suppressor T cells against syngeneic tumors in the mouse.

Splenic T cells from animals bearing growing syngeneic tumors specifically inhibited the effector process of tumor cell lysis by the cytotoxic T cell which had been activated in vitro by mitomycin C treated homologous tumor. The suppression was strictly specific for the individual tumor by which suppressor cells were generated, whereas in some cases cytotoxic T cells generated by two closely related sarcomas showed a certain degree of crossreactivity. This suggests that suppressor and cytotoxic T cells recognize different antigenic moieties on tumor cells; one unique to the individual tumor and the other shared by related tumor cell lines.The suppressor T cell from tumor bearing animals possessed Ia antigen controlled by a gene in I-J subregion of H-2 major histocompatibility complex. Cytotoxic T cells generated by some but not all syngeneic tumors were also killed by anti-Ia and complement; however, the Ia antigen on such cytotoxic T cells was found to be controlled by a locus in I-A subregion. In general, the cytotoxic T cells generated by newly established tumor cell lines had Ia antigen, whereas some old cell lines, which were capable of growing across the H-2 barrier, activated the Ia negative cytotoxic T cell. These results collectively indicate that the immunological resistance against tumors is dependent on the balance of activations of the cytotoxic and suppressor T cells with different specificities and phenotypic expressions.     

Pulsatilla chinensis saponins cause liver injury through interfering ceramide/sphingomyelin balance that promotes lipid metabolism dysregulation and apoptosis

Background: P. chinensis saponins (PRS) are pentacyclic triterpenoid bioactive constituents from Pulsatilla chinensis (Bunge) Regel. In our previous study, PRS caused chronic liver injury (CLI) with the significant changes of lipid metabolites including sphingomyelin (SM) in serum after long-term administration. The SM in the hepatocytes membrane plays an indispensable role in maintaining cell membrane stability and regulating the extracellular and intracellular signal transduction. However, it is still unknown the pathway related to SM and the mechanism of CLI on hepatocyte.Purpose: The purpose of this study was to explore the hepatotoxicity mechanism of PRS in vivo and in vitro, to reveal the action of mechanism of SM and the pathway related to liver injury.Methods: SD rats were orally administered with PRS for 240 days and liver injury was evaluated by histological examinations. Metabolomics analysis was used to explore the liver metabolic pathway affected by PRS, and the expressions of related proteins were evaluated by western blots. To discover and elucidate the underlying mechanisms of metabolites changes induced by PRS at the cellular level, cellular morphology, MTT assays, western blots and cell membrane potential measurements were carried out using LO2 cells. Furthermore, the roles of SM and cholesterol (Chol) in hepatocyte injury were investigated individually in overload Chol and SM groups. Sphingolipid metabolic pathway related with ceramide/sphingomyelin (Cer/SM) balance was explored using cellular lipidomics and RT-PCR.Results: PRS gradually damaged the rat's liver in a time-dependent manner. The analysis of liver metabolism profiles showed that lipids metabolites were changed, including sphingolipid, bile acid, linoleic acid and fatty acid. We found that PRS induced apoptosis by interfering with bile acid-mediated sphingolipid metabolic pathway and Cer/SM balance in CLI. In in vitro experiments, PRS led to the increase of LDH leakage, depolarized cell membrane potential and caused cell membrane toxicity. Furthermore, PRS inducedG0/G1 phase cell cycle arrest in LO2 cells, simultaneously activated cellular extrinsic and intrinsic apoptosis pathways. PRS acted on SM and interfered with Cer/SM balance, which promote lipid metabolism dysregulation and apoptosis.Conclusion: PRS acted on SM to interfere Cer/SM balance on LO2 cell. Both in vivo and in vitro, PRS induced Cer/SM imbalance which promoted lipid metabolism disorder and apoptosis. Apoptosis and lipids changes gradually damaged the rats liver, and ultimately developed into CLI.     

Molecular evolution of the actin-like MreB protein gene family in wall-less bacteria

The mreB gene family encodes actin-like proteins that determine cell shape by directing cell wall synthesis and often exists in one to three copies in the genomes of non-spherical bacteria. Intriguingly, while most wall-less bacteria do not have this gene, five to seven mreB homologs are found in Spiroplasma and Haloplasma, which are both characterized by cell contractility. To investigate the molecular evolution of this gene family in wall-less bacteria, we sampled the available genome sequences from these two genera and other related lineages for comparative analysis. The gene phylogenies indicated that the mreB homologs in Haloplasma are more closely related to those in Firmicutes, whereas those in Spiroplasma form a separate clade. This finding suggests that the gene family expansions in these two lineages are the results of independent ancient duplications. Moreover, the Spiroplasma mreB homologs can be classified into five clades, of which the genomic positions are largely conserved. The inference of gene gains and losses suggests that there has been an overall trend to retain only one homolog from each of the five mreB clades in the evolutionary history of Spiroplasma.     

Cell morphology, ultrastructure, and calcification pattern of Oocardium stratum, a peculiar lotic desmid

Cell morphology and ultrastructure of the desmid Oocardium stratum and its habitat conditions in two limestone-precipitating spring habitats in the Alps were studied. In spite of specific cell geometry, we found ultrastructural features (nucleus with nucleolus, Golgi apparatus, chloroplast structure, lipid bodies, cell wall texture) closely related to other desmids. The type of the mucilage pore apparatus perforating in high densities extended areas of the cell wall of Oocardium is of the Cosmarium type. Oocardium contrasts to Cosmarium by a peculiar bilateral cell geometry (lateral sphenoid shape) which is combined with a dislocated nucleus. Although the cell features of Oocardium did not differ between the two habitats, different calcification types (rhombohedral calcite versus fascicular-fibrous calcite) and calcification intensities were recorded. The spatial positioning and extension of the Oocardium niches differed considerably between the two springs in spite of high CO₂ oversaturation at both sites.     

Characterization of Pneumocystis carinii PHR1, a pH-Regulated Gene Important for Cell Wall Integrity

Pneumocystis carinii remains an important opportunistic fungal pathogen causing life-threatening pneumonia in patients with AIDS and malignancy. Currently, little is known about how the organism adapts to environmental stresses and maintains its cellular integrity. We recently discovered an open reading frame approximately 600 bp downstream of the region coding GSC-1, a gene mediating β-glucan cell wall synthesis in P. carinii. The predicted amino acid sequence of this new gene, termed P. carinii PHR1, exhibited 38% homology to Saccharomyces cerevisiae GAS1, a glycosylphosphatidylinositol-anchored protein essential to maintaining cell wall integrity, and 37% homology to Candida albicans PHR1/PHR2, pH-responsive genes encoding proteins recently implicated in cross-linking β-1,3- and β-1,6-glucans. In view of its homology to these related fungal genes, the pH-dependent expression of P. carinii PHR1 was examined. As in C. albicans, P. carinii PHR1 expression was repressed under acidic conditions but induced at neutral and more alkaline pH. PHR1-related proteins have been implicated in glucan cell wall stability under various environmental conditions. Although difficulties with P. carinii culture and transformation have traditionally limited assessment of gene function in the organism itself, we have successfully used heterologous expression of P. carinii genes in related fungi to address functional correlates of P. carinii-encoded proteins. Therefore, the potential role of P. carinii PHR1 in cell wall integrity was examined by assessing its ability to rescue an S. cerevisiae gas1 mutant with absent endogenous Phr1p-like activity. Interestingly, P. carinii PHR1 DNA successfully restored proliferation of S. cerevisiae gas1 mutants under lethal conditions of cell wall stress. These results indicate that P. carinii PHR1 encodes a protein responsive to environmental pH and capable of mediating fungal cell wall integrity.     

Contrasting carbon metabolism in saprotrophic and pathogenic microascalean fungi from Protea trees

Protea-associated Knoxdaviesia species grow on decaying inflorescences, yet are closely related to plant pathogens such as Ceratocystis albifundus. C. albifundus also infects Protea, but occupies a distinct niche. We investigated substrate utilization in two Knoxdaviesia saprotrophs, a generalist and a specialist, and the pathogen C. albifundus by integrating phenome and whole-genome data. On shared substrates, the generalist grew slightly better than its specialist counterpart, alluding to how it has maintained its Protea host range. C. albifundus grew on few substrates and had limited cell wall-degrading enzymes. It did not utilize sucrose, but may prefer soluble oligosaccharides. Nectar monosaccharides are likely important carbon sources for early colonizing Knoxdaviesia species. Once the inflorescence ages, they could switch to degrading cell wall components. C. albifundus likely uses its limited cell wall-degrading arsenal to gain access to plant cells and exploit internal resources. Overall, carbon metabolism and gene content in three related fungi reflected their ecological adaptations.     

Extreme Divergence of Wolbachia Tropism for the Stem-Cell-Niche in the Drosophila Testis

Microbial tropism, the infection of specific cells and tissues by a microorganism, is a fundamental aspect of host-microbe interactions. The intracellular bacteria Wolbachia have a peculiar tropism for the stem cell niches in the Drosophila ovary, the microenvironments that support the cells producing the eggs. The molecular underpinnings of Wolbachia stem cell niche tropism are unknown. We have previously shown that the patterns of tropism in the ovary show a high degree of conservation across the Wolbachia lineage, with closely related Wolbachia strains usually displaying the same pattern of stem cell niche tropism. It has also been shown that tropism to these structures in the ovary facilitates both vertical and horizontal transmission, providing a strong selective pressure towards evolutionary conservation of tropism. Here we show great disparity in the evolutionary conservation and underlying mechanisms of stem cell niche tropism between male and female gonads. In contrast to females, niche tropism in the male testis is not pervasive, present in only 45% of niches analyzed. The patterns of niche tropism in the testis are not evolutionarily maintained across the Wolbachia lineage, unlike what was shown in the females. Furthermore, hub tropism does not correlate with cytoplasmic incompatibility, a Wolbachia-driven phenotype imprinted during spermatogenesis. Towards identifying the molecular mechanism of hub tropism, we performed hybrid analyses of Wolbachia strains in non-native hosts. These results indicate that both Wolbachia and host derived factors play a role in the targeting of the stem cell niche in the testis. Surprisingly, even closely related Wolbachia strains in Drosophila melanogaster, derived from a single ancestor only 8,000 years ago, have significantly different tropisms to the hub, highlighting that stem cell niche tropism is rapidly diverging in males. These findings provide a powerful system to investigate the mechanisms and evolution of microbial tissue tropism.     

Identification of abnormally high expression of POGZ as a new biomarker associated with a poor prognosis in osteosarcoma

Osteosarcoma (OS) is the most prevalent malignant bone tumor in children and young adults. There is an urgent need for a novel biomarker related to the prognosis of OS. We performed a meta-analysis incorporating six independent datasets and performed a survival analysis with one independent dataset {"type":"entrez-geo","attrs":{"text":"GSE21257","term_id":"21257"}}GSE21257 in the GEO database for gene screening. The results revealed that one potential biomarker related to OS survival, POGZ, was the most significantly upregulated gene. We also verified that the POGZ was overexpressed in clinical samples. The survival analysis revealed that POGZ is associated with a poor prognosis in OS. Moreover, flow cytometry analysis of isolated OS cells demonstrated that OS cells were arrested in the G¹ phase after POGZ knockdown. The RNA-seq results indicated that POGZ was co-expressed with CCNE1 and CCNB1. Pathway analysis showed that genes associated with high expression levels of POGZ were related to the cell cycle pathway. A cell model was constructed to detect the effects of POGZ. After POGZ knockdown, OS cell proliferation, invasion and migration were all decreased. Therefore, POGZ is an important gene for evaluating the prognosis of OS patients and is a potential therapeutic target.Key words: Prognosis, osteosarcoma, biomarkers, POGZ, cell cycle     

Evidence that Tn5565, which includes the enterotoxin gene in Clostridium perfringens, can have a circular form which may be a transposition intermediate

The Clostridium perfringens enterotoxin gene is on a transposon-like element, Tn5565, integrated in the chromosome in human food poisoning strains. The flanking IS elements, IS1470 A and B, are related to IS30. The IS element found in the transposon, IS1469, is related to IS200 and has been found upstream of cpe in all Type A strains. PCR and sequencing studies from cell extracts and plasmid isolations of C. perfringens indicate that Tn5565 can form a circular form with the tandem repeat (IS1470)₂, similar to the transposition intermediates described for a number of IS elements.     

Cell wall proteins at low water potentials

We investigated the proteins extractable from cell walls of stem tissues when plants were subjected to low water potentials (low ψ_w). Dark-grown soybean seedlings (Glycine max [L.] Merr.) showed decreased stem growth when the roots were exposed to vermiculite having low water content (ψ_w = −3 bar). After a time, growth resumed but at a reduced rate relative to the controls. The extractable protein increased in the cell walls as ψ_w decreased, especially a 28-kilodalton protein in the young tissue. In contrast, a 70 kilodalton protein, mainly extractable from mature cell walls, appeared to decrease slightly at low ψ_w. No hydroxyproline was present in either protein, which shows that neither protein is related to extensin. The level of the 28 kilodalton protein increased in the cell wall of the dividing region soon after the initial growth inhibition, and it appeared in the elongating tissue at about the time growth resumed. The correlation between growth and these protein changes suggests that the two events could be related.     

Function of the aux and rol genes of the Ri plasmid in plant cell division in vitro

Auxin-autonomous growth in vitro may be related to the integration and expression of the aux and rol genes from the root-inducing (Ri) plasmid in plant cells infected by agropine-type Agrobacterium rhizogenes. To elucidate the functions of the aux and rol genes in plant cell division, plant cell lines transformed with the aux1 and aux2 genes or with the rolABCD genes were established using tobacco (Nicotiana tabacum) Bright Yellow-2 (BY-2) cells. The introduction of the aux1 and aux2 genes enabled the auxin-autonomous growth of BY-2 cells, but the introduction of the rolABCD genes did not affect the auxin requirement of the BY-2 cells. The results clearly show that the aux genes are necessary for auxinautotrophic cell division, and that the rolABCD genes are irrelevant in auxin autotrophy.Key words: Agrobacterium rhizogenes, auxin-autotrophic cell, auxin biosynthesis, hairy root, plant cell division, Ri plasmid, T-DNA, aux, rol, tobacco BY-2 cells     

Effects of nemorosone,isolated from the plant Clusia rosea,on the cell cycle and gene expression in MCF-7 BUS breast cancer cell lines

Background: Breast cancer is the cause of considerable morbidity and mortality in women. While estrogen receptor antagonists have been widely used in breast cancer treatment, patients have increasingly shown resistance to these agents and the identification of novel targeted therapies is therefore required. Nemorosone is the major constituent of the floral resin from Clusia rosea and belongs to the class of polycyclic polyisoprenylated benzophenones of the acylphloroglucinol group. The cytotoxicity of nemorosone in human cancer cell lines has been reported in recent years and has been related to estrogen receptors in breast cancer cells.Methods: Changes induced by nemorosone in the cell cycle and gene expression of the MCF-7 BUS (estrogen-dependent) breast cancer cell line were analyzed using flow cytometry and the RT² Profiler PCR array, respectively.Results: In comparison to breast cancer cells without treatment, nemorosone induced discrete cell cycle arrest in the G1 phase and significant depletion in the G2 phase. Moreover, the compound altered the expression of 19 genes related to different pathways, especially the cell cycle, apoptosis and hormone receptors.Conclusion: These promising results justify further studies to clarify mechanisms of action of nemorosone, in view of evaluate the possible use of this benzophenone as adjuvant in the treatment of breast cancer.     

Exosomes Secreted by Toxoplasma gondii-Infected L6 Cells: Their Effects on Host Cell Proliferation and Cell Cycle Changes

Toxoplasma gondii infection induces alteration of the host cell cycle and cell proliferation. These changes are not only seen in directly invaded host cells but also in neighboring cells. We tried to identify whether this alteration can be mediated by exosomes secreted by T. gondii-infected host cells. L6 cells, a rat myoblast cell line, and RH strain of T. gondii were selected for this study. L6 cells were infected with or without T. gondii to isolate exosomes. The cellular growth patterns were identified by cell counting with trypan blue under confocal microscopy, and cell cycle changes were investigated by flow cytometry. L6 cells infected with T. gondii showed decreased proliferation compared to uninfected L6 cells and revealed a tendency to stay at S or G2/M cell phase. The treatment of exosomes isolated from T. gondii-infected cells showed attenuation of cell proliferation and slight enhancement of S phase in L6 cells. The cell cycle alteration was not as obvious as reduction of the cell proliferation by the exosome treatment. These changes were transient and disappeared at 48 hr after the exosome treatment. Microarray analysis and web-based tools indicated that various exosomal miRNAs were crucial for the regulation of target genes related to cell proliferation. Collectively, our study demonstrated that the exosomes originating from T. gondii could change the host cell proliferation and alter the host cell cycle.