Integrating S-phase checkpoint signaling with trans-lesion synthesis of bulky DNA adducts

Bulky adducts are DNA lesions generated in response to environmental agents including benzo[a]pyrene (a combustion product) and solar ultraviolet radiation. Error-prone replication of adducted DNA can cause mutations, which may result in cancer. To minimize the detrimental effects of bulky adducts and other DNA lesions, S-phase checkpoint mechanisms sense DNA damage and integrate DNA repair with ongoing DNA replication. The essential protein kinase Chk1 mediates the S-phase checkpoint, inhibiting initiation of new DNA synthesis and promoting stabilization and recovery of stalled replication forks. Here we review the mechanisms by which Chk1 is activated in response to bulky adducts and potential mechanisms by which Chk1 signaling inhibits the initiation stage of DNA synthesis. Additionally, we discuss mechanisms by which Chk1 signaling facilitates bypass of bulky lesions by specialized Y-family DNA polymerases, thereby attenuating checkpoint signaling and allowing resumption of normal cell cycle progression.     

Rice bran lipase catalyzed esterification of palm oil fatty acid distillate and glycerol in organic solvent

Rice bran lipase (RBL) was delipidated to enhance its stability in organic solvent and its esterification activity at elevated temperature. The esterification activity of delipidated RBL increased as temperature was increased from 45 to 65°C. The esterification activity of delipidated RBL at 65°C was about 14 times greater than that of the non-delipidated RBL. As temperature was further increased to 75°C, the non-delipidated RBL lost all esterification activity, whereas the delipidated RBL retained approximately 48% of its esterilication activity. The delipidated RBL maintained a relative esterification activity greater than 80% after 16 h of incubation in hexane, whereas the non-delipidated RBL maintained a relative esterification activity of only 50%. A method for production of acylglycerol using delipidated RBL to esterify palm oil fatty acid distillate (PFAD) with glycerol in hexane was successfully developed. The effects of reaction temperatures and type of water removal agents (silica gel and molecular sieve) on the degree of esterification were also examined. A 4 h reaction at 65°C, catalyzed by delipidated RBL and using silica gel as the water removal agent resulted in 53.8% esterification. Thin layer chromatography analysis suggested that the esterified product was primarily comprised of mono-and di-acylglycerols.     

Synthesis of Aβ[1‐42] and its derivatives with improved efficiency

It has been proved that the principal component of senile plaques is aggregates of β‐amyloid peptide (Aβ) in cases of one of the most common forms of age‐related neurodegenerative disorders, Alzheimer's disease (AD). Although the synthetic methods for the synthesis of Aβ peptides have been developed since their first syntheses, Aβ[1‐42] is still problematic to prepare. The highly hydrophobic composition of Aβ[1‐42] results in aggregation between resin‐bound peptide chains or intrachain aggregation which leads to a decrease in the rates of deprotection and repetitive incomplete coupling reactions during 9‐flurenylmethoxycarbonyl (Fmoc) synthesis. In order to avoid aggregation and/or disrupt internal aggregation during stepwise Fmoc solid phase synthesis and to improve the quality of crude products, several attempts have been made. Since highly pure Aβ peptides in large quantities are used in biological experiments, we wanted to develop a method for a rational synthesis of human Aβ[1‐42] with high purity and adequate yield. This paper reports a convenient methodology with a novel solvent system for the synthesis of Aβ[1‐42], its N‐terminally truncated derivatives Aβ[4‐42] and Aβ[5‐42], and Aβ[1‐42] labeled with 7‐amino‐4‐methyl‐3‐coumarinylacetic acid (AMCA) at the N‐terminus using Fmoc strategy. The use of 10% anisole in Dimethylformamide/Dichloromethane (DMF/DCM) can substantially improve the purity and yield of crude Aβ[1‐42] and has been shown to be an optimal coupling condition for the synthesis of Aβ[1‐42]. Anisole is a cheap and simple aid in the synthesis of ‘difficult sequences’ where other solvents are less successful in the prevention of aggregation during the synthesis. Copyright © 2006 European Peptide Society and John Wiley & Sons, Ltd.     

An Improved Method of Gene Synthesis Based on DNA Works Software and Overlap Extension PCR

A bottleneck in recent gene synthesis technologies is the high cost of oligonucleotide synthesis and post-synthesis sequencing. In this article, a simple and rapid method for low-cost gene synthesis technology was developed based on DNAWorks program and an improved single-step overlap extension PCR (OE-PCR). This method enables any DNA sequence to be synthesized with few errors, then any mutated sites could be corrected by site-specific mutagenesis technology or PCR amplification-assembly method, which can amplify different DNA fragments of target gene followed by assembly into an entire gene through their overlapped region. Eventually, full-length DNA sequence without error was obtained via this novel method. Our method is simple, rapid and low-cost, and also easily amenable to automation based on a DNAWorks design program and defined set of OE-PCR reaction conditions suitable for different genes. Using this method, several genes including Manganese peroxidase gene (Mnp) of Phanerochaete chrysosporium (P. chrysosporium), Laccase gene (Lac) of Trametes versicolor (T. versicolor) and Cip1 peroxidase gene (cip 1) of Coprinus cinereus (C. cinereus) with sizes ranging from 1.0 kb to 1.5 kb have been synthesized successfully. Bingxue Dong and Runqian Mao contributed equally to this work.     

Gregarina niphandrodes may lack both a plastid genome and organelle

Gregarines are early diverging apicomplexans that appear to be closely related to Cryptosporidium. Most apicomplexans, including Plasmodium, Toxoplasma, and Eimeria, possess both plastids and corresponding plastid genomes. Cryptosporidium lacks both the organelle and the genome. To investigate the evolutionary history of plastids in the Apicomplexa, we tried to determine whether gregarines possess a plastid and/or its genome. We used PCR and dot-blot hybridization to determine whether the gregarine Gregarina niphandrodes possesses a plastid genome. We used an inhibitor of plastid function for any reduction in gregarine infection, and transmission electron microscopy to search for plastid ultrastructure. Despite an extensive search, an organelle of the appropriate ultrastructure in transmission electron microscopy, was not observed. Triclosan, an inhibitor of the plastid-specific enoyl-acyl carrier reductase enzyme, did not reduce host infection by G. niphandrodes. Plastid-specific primers produced amplicons with the DNA of Babesia equi, Plasmodium falciparum, and Toxoplasma gondii as templates, but not with G. niphandrodes DNA. Plastid-specific DNA probes, which hybridized to Babesia equi, failed to hybridize to G. niphandrodes DNA. This evidence indicates that G. niphandrodes is not likely to possess either a plastid organelle or its genome. This raises the possibility that the plastid was lost in the Apicomplexan following the divergence of gregarines and Cryptosporidium.     

Preapoptotic chromatin changes induced by ultraviolet B irradiation in human erythroleukemia K562 cells

Exponentially growing human erythroleukemia K562 cells were permeabilized and the dose dependent decrease of DNA synthesis rate was measured after ultraviolet (UV B, 290 nm) irradiation. Cells were able to overcome 2 and 5 J/m2 UV doses, partial recovery was observed at 15 J/m2, while at high (25 J/m2) UV dose replicative DNA synthesis remained suppressed. K562 cells were subjected to synchronization prior to and after UV irradiation (24 J/m2) and 18 fractions were collected by centrifugal elutriation. Cell cycle analysis by flow cytometry did not show early apoptotic cells after UV irradiation. The gradual increase in DNA content typical for non-irradiated cells was contrasted by an early S phase block between 2.2 and 2.4 C-values after UV irradiation. Cell cycle dependent chromatin changes after ultraviolet irradiation were seen as a fine fibrillary network covering the mainly fibrous chromatin structures and incompletely folded primitive chromosomes. Based on observations after UV irradiation and on earlier results with cadmium treatment and gamma irradiation, we confirm that typical chromatin changes characteristic to genotoxic agents can be recognized and classified.     

Microscopical investigation of poly(3-hydroxybutyrate) granule formation in Azotobacter vinelandii

Poly(3-hydroxybutyrate) (PHB) granule formation in Azotobacter vinelandii was investigated by laser scanning fluorescence microscopy after staining the cells with Nilered and Baclight. Cells that had been starved for a carbon source for > or =3 days were almost free of PHB granules. Formation of visible PHB granules started within 1-2 h after transfer of the cells to a medium permissive for PHB accumulation. Fluorescent PHB granules at the early stages of formation were exclusively found in the cell periphery of the 2-3 mum ovoid-shaped cells. After 3 h of PHB accumulation or later, PHB granules were also found to be detached from the cell periphery. Our results indicate that PHB granule formation apparently begins at the inner site of the cytoplasmic membrane. This finding is different from previous assumptions that PHB granule formation occurs randomly in the cytoplasm of PHB-accumulating bacteria.     

Sequence analysis of the 181-kb accessory plasmid pSmeSM11b, isolated from a dominant Sinorhizobium meliloti strain identified during a long-term field release experiment

The 181 251 bp accessory plasmid pSmeSM11b of Sinorhizobium meliloti strain SM11, belonging to a dominant indigenous S. meliloti subpopulation identified during a long-term field release experiment, was sequenced. This plasmid has 166 coding sequences (CDSs), 42% of which encode proteins with homology to proteins of known function. Plasmid pSmeSM11b is a member of the repABC replicon family and contains a large gene region coding for a conjugation system similar to that of other self-transmissible plasmids in Rhizobium and Agrobacterium. Another pSmeSM11b gene region, possibly involved in sugar metabolism and polysaccharide catabolism, resembled a region of S. meliloti 1021 megaplasmid pSymB and in the genome of Sinorhizobium medicae WSM419. Another module of plasmid pSmeSM11b encodes proteins similar to those of the nitrogen-fixing actinomycete Frankia CcI3, and which are likely to be involved in the synthesis of a secondary metabolite. Several ORFs of pSmeSM11b were predicted to play a role in nonribosomal peptide synthesis. Plasmid pSmeSM11b has many mobile genetic elements, which contribute to the mosaic composition of the plasmid.