Embryonic stem cells lacking the epigenetic regulator Cfp1 are hypersensitive to DNA-damaging agents and exhibit decreased Ape1/Ref-1 protein expression and endonuclease activity

Modulation of chromatin structure plays an important role in the recruitment and function of DNA repair proteins. CXXC finger protein 1 (Cfp1), encoded by the CXXC1 gene, is essential for mammalian development and is an important regulator of chromatin structure. Murine embryonic stem (ES) cells lacking Cfp1 (CXXC1^?/?) are viable but demonstrate a dramatic decrease in cytosine methylation, altered histone methylation, and an inability to differentiate. We find that ES cells lacking Cfp1 are hypersensitive to a variety of DNA-damaging agents. In addition, CXXC1^?/? ES cells accumulate more DNA damage and exhibit decreased protein expression and endonuclease activity of AP endonuclease (Ape1/Ref-1), an enzyme involved in DNA base excision repair. Expression in CXXC1^?/? ES cells of either the amino half of Cfp1 (amino acids 1–367) or the carboxyl half of Cfp1 (amino acids 361–656) restores normal Ape1/Ref-1 protein expression and rescues the hypersensitivity to DNA-damaging agents, demonstrating that Cfp1 contains redundant functional domains. Furthermore, retention of either the DNA-binding activity of Cfp1 or interaction with the Setd1A and Setd1B histone H3-Lys4 methyltransferase complexes is required to restore normal sensitivity of CXXC1^?/? ES cells to DNA-damaging agents. These results implicate Cfp1 as a regulator of DNA repair processes.     

The Epigenetic Regulator CXXC Finger Protein 1 is Essential for Murine Hematopoiesis

CXXC finger protein 1 (Cfp1), encoded by the Cxxc1 gene, binds to DNA sequences containing an unmethylated CpG dinucleotide and is an epigenetic regulator of both cytosine and histone methylation. Cxxc1-null mouse embryos fail to gastrulate, and Cxxc1-null embryonic stem cells are viable but cannot differentiate, suggesting that Cfp1 is required for chromatin remodeling associated with stem cell differentiation and embryogenesis. Mice homozygous for a conditional Cxxc1 deletion allele and carrying the inducible Mx1-Cre transgene were generated to assess Cfp1 function in adult animals. Induction of Cre expression in adult animals led to Cfp1 depletion in hematopoietic cells, a failure of hematopoiesis with a nearly complete loss of lineage-committed progenitors and mature cells, elevated levels of apoptosis, and death within two weeks. A similar pathology resulted following transplantation of conditional Cxxc1 bone marrow cells into wild type recipients, demonstrating this phenotype is intrinsic to Cfp1 function within bone marrow cells. Remarkably, the Lin⁻Sca-1⁺c-Kit⁺ population of cells in the bone marrow, which is enriched for hematopoietic stem cells and multi-potential progenitor cells, persists and expands in the absence of Cfp1 during this time frame. Thus, Cfp1 is necessary for hematopoietic stem and multi-potential progenitor cell function and for the developmental potential of differentiating hematopoietic cells.     

Cytotoxicity responses to selected ESAT-6 and CFP-10 peptides in tuberculosis

Cytotoxicity responses were studied for the ESAT-6 peptides Esp1, Esp6, Esp7, Esp8, and CFP-10 peptides, Cfp6, Cfp7, Cfp8, Cfp9 (synthetic 20-mer peptides) and the recombinant ESAT-6, CFP-10 proteins. Cytolytic molecules perforin, granzymes A and B, granulysin responses in healthy household contacts (HHC) and pulmonary tuberculosis patients (PTB), were studied by intracellular flow cytometry. Functional cytotoxicity was studied in both the groups for the peptides Esp6 and Cfp8 by an enzyme (lactate dehydrogenase) based assay. The results revealed that cytolytic molecule positive CD4+ and CD8+ T cells were increased in HHC in response to Esp1, Esp6, Cfp8 and Cfp9 immunogenic peptides compared to PTB. Functional cytotoxicity results showed higher cytotoxicity (not statistically significant) to be exhibited by the peptide Esp6 than Cfp8 in the HHC.     

Changes in ribosomal proteins during colony development in Streptomyces.

The structure and functionality of the ribosomal subunits of the substrate and the aerial mycelium of Streptomyces antibioticus were compared. Using SDS-PAGE and HPLC, several differences between the ribosomal protein pattern from both stages of development were observed, including a clear decrease in the L7/L12 content of the aerial mycelium. The activity of the aerial mycelia ribosomes was also decreased when compared with that of the substrate mycelium. This effect was more pronounced in the 50S subunit. These results suggest that during cell differentiation in Streptomyces important changes occur at the ribosomal level, particularly in the transition from the substrate to the aerial mycelium.     

Receptor/ligand interactions between Cryptosporidium parvum and the surface of the host cell.

The ability of membrane antigens on sporozoites of the intestinal pathogen, Cryptosporidium parvum, to bind host cell surface antigens was investigated. A novel membrane-associated protein of approximately 47 kDa, designated CP47, was found to possess significant binding affinity for the surface of both human and animal ileal cells. This protein was purified by a combination of anion-exchange chromatography on FPLC and immunoaffinity chromatography. Purified CP47 demonstrated competitive binding with parasite-associated membrane antigens to membranes of HCT-8 and ileal cells in a dose-dependent manner. Furthermore, the binding activity of CP47 was found to be Mn2+-sensitive, and was completely inhibited in the presence of 10 mM MnCl2. These results were consistent with earlier findings demonstrating the inhibitory effect of Mn2+ ions on Cryptosporidium infection both in vitro and in vivo (Nesterenko et al., Biol. Trace Elem. Res. 56 (1997) 243-253). Immunoelectron microscopy using gold-conjugated antibodies revealed CP47 to be localized at the apical end of the sporozoites. A single protein with an electrophoretic mobility of 57 kDa was purified from host cell membranes using CP47-Affigel. Similarly, affinity purification of this protein was abrogated in the presence of Mn2+. These data suggest that a novel parasite protein, CP47, may play an important role in sporozoite/host cell attachment.     

Factors affecting production of mold mycelium and protein in synthetic media.

The effects of certain cultural conditions on the yield of dry mycelium, protein, and total amino acid content of Rhizopus oligosporus Saito (NRRL 2710), Rhizopus rhizopodiformis (Cohn apud Lichtheim) Zopf (NRRL 6246), and Absidia corymbifera (Cohn) Sacc. et Trotter (NRRL 6247) were studied. The yield of mycelium was found to significantly increase as the spore inoculum was increased from 187,500 to 2,250,000 spores. But the total amino acids (grams/liter) did not change significantly, whereas the percentage of crude protein decreased. An inoculum containing approximately 750,000 spores/ml was used in all of the other experiments. Mycelial production was highest at 37 degrees C for all three molds. However, the best temperature for percentage of crude protein and total amino acids varied with the organism. The mycelial yield and total crude protein of R. oligosporus showed some significant changes as the C/N ratio was increased in 3% glucose medium. In a synthetic medium having a 15:1 C/N ratio, the strains of R. oligosporus, R. rhizopodiformis, and A. corymbifera had better yields from falactose than glucose, not only in dry mycelium but also in total crude protein (grams/liter) and total amino acids (grams/liter). R. oligosporus grew very well on several ammonium salts. but the maximum yield of dry mycelium, total crude protein (grams/liter), and total amino acids (grams/liter) occurred with ammonium sulfate. The optimum pH for both Rhizopus species was 4.0, although R. oligosporus grew equally well at pH 3.0 and slightly less at pH 5.0. The highest yield of mycelium for A. corymbifera was obtained in a medium with an initial pH of 8.0. It was calculated that a fermenter chanrged with an adequate medium and 1,000 lb (about 450 kg) of R. oligosporus or A. corymbifera cells could produce 88 or 90 lb of protein (on a dry-weight basis) per h if the product was removed continuously.     

Factors affecting production of mold mycelium and protein in synthetic media.

The effects of certain cultural conditions on the yield of dry mycelium, protein, and total amino acid content of Rhizopus oligosporus Saito (NRRL 2710), Rhizopus rhizopodiformis (Cohn apud Lichtheim) Zopf (NRRL 6246), and Absidia corymbifera (Cohn) Sacc. et Trotter (NRRL 6247) were studied. The yield of mycelium was found to significantly increase as the spore inoculum was increased from 187,500 to 2,250,000 spores. But the total amino acids (grams/liter) did not change significantly, whereas the percentage of crude protein decreased. An inoculum containing approximately 750,000 spores/ml was used in all of the other experiments. Mycelial production was highest at 37 degrees C for all three molds. However, the best temperature for percentage of crude protein and total amino acids varied with the organism. The mycelial yield and total crude protein of R. oligosporus showed some significant changes as the C/N ratio was increased in 3% glucose medium. In a synthetic medium having a 15:1 C/N ratio, the strains of R. oligosporus, R. rhizopodiformis, and A. corymbifera had better yields from falactose than glucose, not only in dry mycelium but also in total crude protein (grams/liter) and total amino acids (grams/liter). R. oligosporus grew very well on several ammonium salts. but the maximum yield of dry mycelium, total crude protein (grams/liter), and total amino acids (grams/liter) occurred with ammonium sulfate. The optimum pH for both Rhizopus species was 4.0, although R. oligosporus grew equally well at pH 3.0 and slightly less at pH 5.0. The highest yield of mycelium for A. corymbifera was obtained in a medium with an initial pH of 8.0. It was calculated that a fermenter chanrged with an adequate medium and 1,000 lb (about 450 kg) of R. oligosporus or A. corymbifera cells could produce 88 or 90 lb of protein (on a dry-weight basis) per h if the product was removed continuously.     

cDNA sequence analysis of CP94: rat lens fiber cell beaded-filament structural protein shows homology to cytokeratins.

To study the molecular structure of the gene responsible for a lens fiber cell beaded-filament structural protein of 94kDa (CP94), we isolated its specific cDNA from a rat lens cDNA library by use of anti-mouse CP94 antiserum. The expressed fusion protein kept the epitopes specific against anti-chick CP97 as well as anti-mouse CP94 antibody, and the size was estimated as 190-200kDa, indicating that the cDNA insert of the clone seemed to encode a polypeptide with 80-90kDa in appearance. Northern analysis indicated that CP94 mRNA is expressed only in the lens, and not in the brain, skin, heart, kidney, lung, and liver, and the size was estimated to 2.1-2.3kb. In a lens of inherited microphthalmic mouse, Elo, a trace amount of mRNA with the size closely similar to that of rat mRNA was observed. The entire compiled sequence (1,873bp) showed an open reading frame covering the sequence of 533 amino acids totalling 58,857Da. No sequence homologous to the entire CP94 was found among the entries of any nucleotide and amino acid sequence databases; but with respect to a limited amino acid sequence of N-side region of CP94, a significant homology with cytokeratins was found.     

采用SCGE和SCE分析法研究姬松茸菌体多糖(Ab-Mp)对DNA损伤的抑制作用

热水浸提法提取姬松茸菌丝体多糖,每天以10mg/(kg bw)和20mg/(kg bw)两种剂量对小鼠进行灌胃,连续15d.腹腔注射环磷酰胺(CP),运用单细胞凝胶电泳(SCGE)法和姐妹染色体交换(SCE)分析法,研究Ab-Mp对染色体损伤的保护作用.结果表明,Ab-Mp降低了CP损伤后彗星细胞的比例,缩短了彗星尾长;降低了CP损伤的姐妹染色单体交换率.Ab-Mp对环磷酰胺诱发的DNA损伤具有拮抗作用.     

Continuous fermentation to produce fungal protein. Effect of growth rate on the biomass yield and chemical composition of Fusarium moniliforme

Fusarium moniliforme was grown on a carob aqueous extract in a chemostat for fungal protein production. The substrate was adjusted to provide 0.5% carob sugars supplemented with inorganic salts. The dilution rate varied from 0.086 to 0.227 hr^?1 under constant conditions of temperature (30°C), pH (4.5), and oxygen saturation (60–80%). A yield of 0.709 g dry mycelium/g consumed carob sugar and a productivity value of 0.687 g dry mycelium/liter hr^?1 were obtained at μ = 0.205 hr^?1. The maintenance coefficient was 0.077 g carob sugar/g dry mycelium hr^?1. While the carbohydrate and purine content of dry mycelium increased at μ values from 0.114 to 0.205 hr^?1 both true (Lowry) and crude (N × 6.25) protein contents decreased at the same μ range. Maximum values of 36.3% true and 47.9% crude protein of dry mycelium were obtained at μ = 0.114 hr^?1, whereas a minimum purine content of 99.8 μmol/g corresponding to 6.42% nucleic acids was recorded at μ = 0.086 hr^?1. It was concluded that a continuous fermentation of carob aqueous extract using F. moniliforme should be operated at growth rates of approximately 0.205 hr^?1 in order to maximize protein production.     

Growth of Fusarium moniliforme on carob aqueous extract and nutritional evaluation of its biomass.

Fusarium moniliforme was cultured semicontinuously on a carob medium in a 14-liter fermentor (8.5-liter working volume). The growth medium provided 2.4% carob sugar, 0.72% NH4H2PO4, and 0.03% MgSO4-7H2O. The biomass harvest was 8.8 g/liter per day. Ninety percent of the sugars were consumed, and the pH dropped from 5.9 to about 3.7. The crude protein (N X 6.25) of the spray-dried mycelium was 380 g/kg, 300 g/kg for the true protein (Lowry), and 4.8 g/kg for the (Folin-Denis) tannic acid. The mycelium was evaluated nutritionally with the weanling rat as experimental animal. The protein efficiency ratio and net protein utilization values for the unsupplemented mycelium were 1.15 and 0.42, respectively, and for the mycelium supplemented with DL-methionine (5 g/kg) they were 2.31 and 0.72, respectively. No growth depression was observed in the experimental rats, and on dissection of the carcasses the internal organs were found to be normal.     

Growth of Fusarium moniliforme on carob aqueous extract and nutritional evaluation of its biomass.

Fusarium moniliforme was cultured semicontinuously on a carob medium in a 14-liter fermentor (8.5-liter working volume). The growth medium provided 2.4% carob sugar, 0.72% NH4H2PO4, and 0.03% MgSO4-7H2O. The biomass harvest was 8.8 g/liter per day. Ninety percent of the sugars were consumed, and the pH dropped from 5.9 to about 3.7. The crude protein (N X 6.25) of the spray-dried mycelium was 380 g/kg, 300 g/kg for the true protein (Lowry), and 4.8 g/kg for the (Folin-Denis) tannic acid. The mycelium was evaluated nutritionally with the weanling rat as experimental animal. The protein efficiency ratio and net protein utilization values for the unsupplemented mycelium were 1.15 and 0.42, respectively, and for the mycelium supplemented with DL-methionine (5 g/kg) they were 2.31 and 0.72, respectively. No growth depression was observed in the experimental rats, and on dissection of the carcasses the internal organs were found to be normal.     

Oxidative modification of human ceruloplasmin by peroxyl radicals.

Ceruloplasmin (CP), the blue oxidase present in all vertebrates, is the major copper-containing protein of plasma. We investigated oxidative modification of human CP by peroxyl radicals generated in a solution containing 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH). When CP was incubated with AAPH, the aggregation of proteins was increased in a time- and dose-dependent manner. Incubation of CP with AAPH resulted in a loss of ferroxidase activity. Superoxide dismutase and catalase did not protect the aggregation of CP, whereas hydroxyl radical scavengers such as ethanol and mannitol protected the protein aggregation. The aggregation of proteins was significantly inhibited by the copper chelators, diethyldithiocarbamate and penicillamine. Exposure of CP to AAPH led to the release of copper ions from the enzyme and the generation of protein carbonyl derivatives. Subsequently, when the amino acid composition of CP reacted with AAPH was analyzed, cysteine, tryptophan, methionine, histidine, tyrosine, and lysine residues were particularly sensitive.     

Cloning and characterization of the A-factor receptor gene from Streptomyces griseus.

A-factor (2-isocapryloyl-3R-hydroxymethyl-gamma-butyrolactone) and its specific receptor protein control streptomycin production, streptomycin resistance, and aerial mycelium formation in Streptomyces griseus. The A-factor receptor protein (ArpA) was purified from a cell lysate of S. griseus IFO 13350. The NH2-terminal amino acid sequences of ArpA and lysyl endopeptidase-generated fragments were determined for the purpose of preparing oligonucleotide primers for cloning arpA by the PCR method. The arpA gene cloned in this way directed the synthesis of a protein having A-factor-specific binding activity when expressed in Escherichia coli under the control of the T7 promoter. The arpA gene was thus concluded to encode a 276-amino-acid protein with a calculated molecular mass of 29.1 kDa, as determined by nucleotide sequencing. The A-factor-binding activity was observed with a homodimer of ArpA. The NH2-terminal portion of ArpA contained an alpha-helix-turn-alpha-helix DNA-binding motif that showed great similarity to those of many DNA-binding proteins, which suggests that it exerts its regulatory function for the various phenotypes by directly binding to a certain key gene(s). Although a mutant strain deficient in both the ArpA protein and A-factor production overproduces streptomycin and forms aerial mycelium and spores earlier than the wild-type strain because of repressor-like behavior of ArpA, introduction of arpA into this mutant abolished simultaneously its streptomycin production and aerial mycelium formation. All of these data are consistent with the idea that ArpA acts as a repressor-type regulator for secondary metabolite formation and morphogenesis during the early growth phase and A-factor at a certain critical intracellular concentration releases the derepression, thus leading to the onset of secondary metabolism and aerial mycelium formation. The presence of ArpA-like proteins among Streptomyces spp., as revealed by PCR, together with the presence of A-factor-like compounds, suggests that a hormonal control similar to the A-factor system exists in many species of this genus.     

利用酵母双杂交系统筛选与黄瓜花叶病毒外壳蛋白互作的寄主蛋白

利用酵母双杂交系统,以黄瓜花叶病毒(Cucumber mosaic virus,CMV)的外壳蛋白(coat protein,CP)为诱饵,从番茄叶片c DNA文库中筛选与其互作的蛋白。结果显示,诱饵载体pBT3-SUC-CMV-CP均能在酵母细胞中正确表达,无自激活活性而且对酵母无毒性;通过对酵母双杂交文库的筛选和回转验证,共获得了98个阳性克隆,分别编码67个可能与CMV-CP相互作用的蛋白,分别参与植物防御反应、光合作用、物质转运、信号转导、能量代谢、氨基酸代谢、细胞壁的形态建成、植物的激素代谢等。本研究结果表明,CMV CP可同时调控寄主的多个代谢过程,在CMV的致病过程中有多重功能。     

Rsistance to streptomycin in a producing strain of Streptomyces griseus.

Streptomyces griseus S 104 was sensitive to streptomycin during exponential growth in a medium which, in the subsequent stationary phase, supported production of the antibiotic in yields above 200 mug/ml. When antibiotic production began cultures developed a tolerance toward their lethal metabolite. This was not due to an increase in pH associated with antibiotic production, since pH effects on streptomycin sensitivity in S. griseus were in the reverse direction. However, the degree of tolerance was directly related to the amount of cell material present. Streptomycin production caused no change in the proportion of resistant variants in the population, nor did it cause the severe inhibition of protein synthesis observed in non-producing cultures exposed to the antibiotic. The lack of an effect on protein synthesis is attributed to the absence of streptomycin with in the cytoplasm since soluble extracts from mycelium harvested in the production phase were inactive when bioassayed immediately after cell disruption. However, they developed antibacterial activity rapidly when heated, and more slowly when incubated at 25 degrees C. The addition of phosphatase inhibitors during incubation prevented the appearance of antibiotic activity, and it was concluded that a small amount of streptomycin phosphate is present in the mycelium during antibiotic production. Differences in (14C) streptomycin uptake suggested that the mycelium was appreciably less permeable to the antibiotic in the production phase than during exponential growth. However, a small amount was taken up and much of it was in the soluble fraction of disrupted cells. Bioassays showed that this 14C-labeled antibiotic within the cells had been partially inactivated, suggesting that conversion of streptomycin to an inactive derivative is involved in the mechanism which protects the organism from its metabolite.     

Mycelium cultivation, chemical composition and antitumour activity of a Tolypocladium sp. fungus isolated from wild Cordyceps sinensis

AIMS: To examine and illustrate the morphological characteristics and growth kinetics of Cs-HK1, a Tolypocladium fungus, isolated from wild Cordyceps sinensis in solid and liquid cultures, and the major chemical constituents and antitumour effects of Cs-HK1 mycelium. METHODS AND RESULTS: The Cs-HK1 fungus was isolated from the fruiting body of a wild C. sinensis and identified as a Tolypocladium sp. fungus. It grew rapidly at 22-25 degrees C on a liquid medium containing glucose, yeast extract, peptone and major inorganic salts, with a specific growth rate of 1.1 day(-1), reaching a cell density of 23.0 g dw l(-1) in 7-9 days. Exopolysaccharides accumulated in the liquid culture to about 0.3 g l(-1) glucose equivalent. In comparison with natural C. sinensis, the fungal mycelium had similar contents of protein (11.7-microg) and carbohydrate (654.6-microg) but much higher contents of polysaccharide (244.2 mg vs 129.5 mg), adenosine (1116.8-microg vs 264.6 microg) and cordycepin (65.7 microg vs 20.8 microg) (per gram dry weight). Cyclosporin A, an antibiotic commonly produced by Tolypocladium sp., was also detected from the mycelium extract. The hot water extract of mycelium showed low cytotoxic effect on B16 melanoma cells in culture (about 25% inhibition) but significant antitumour effect in animal tests, causing 50% inhibition of B16 cell-induced tumour growth in mice. CONCLUSIONS: The Tolypocladium sp. fungus, Cs-HK1, can be easily cultivated by liquid fermentation. The mycelium biomass contained the major bioactive compounds of C. sinensis, and the mycelium extract had significant antitumour activity. SIGNIFICANCE AND IMPACT OF THE STUDY: The Cs-HK1 fungus may be a new and promising medicinal fungus and an effective and economical substitute of the wild C. sinensis for health care.     

Expression analysis of PMP22/Gas3 in premalignant and malignant pancreatic lesions.

PMP22 is a structural protein of Schwann cells, but it also influences cell proliferation. In the present study, quantitative RT-PCR (QRT-PCR) and immunohistochemistry were used to determine PMP22 mRNA levels and to localize PMP22 in the normal pancreas (n=20), chronic pancreatitis (CP) (n=22), pancreatic ductal adenocarcinoma (PDAC) (n=31), intraductal papillary mucinous neoplasms (IPMN) (n=9), mucinous cystic tumors (MCN) (n=4), and in a panel of PanIN lesions (n=29). PMP22 mRNA levels were significantly higher in CP (3-fold) and PDAC (2.5-fold), compared to normal pancreatic tissues. PMP22 expression was restricted to nerves in the normal pancreas, while in CP and PDAC PMP22 was also expressed in PanIN lesions and in a small percentage of pancreatic cancer cells. PMP22 was weak to absent in the tumor cells of IPMNs and MCNs. PMP22 mRNA was present at different levels in cultured pancreatic cancer cells and up-regulated by transforming growth factor (TGF)-beta1 in 2 of 8 of these cell lines. In conclusion, PMP22 expression is present in both CP and PDAC tissues. Its expression in PanIN lesions and some pancreatic cancer cells in vitro and in vivo suggests a role of PMP22 in the neoplastic transformation process from the normal pancreas to pre-malignant lesions to pancreatic cancer.