Conditional-ready mouse embryonic stem cell derived macrophages enable the study of essential genes in macrophage function.

The ability to differentiate genetically modified mouse embryonic stem (ES) cells into functional macrophages provides a potentially attractive resource to study host-pathogen interactions without the need for animal experimentation. This is particularly useful in instances where the gene of interest is essential and a knockout mouse is not available. Here we differentiated mouse ES cells into macrophages in vitro and showed, through a combination of flow cytometry, microscopic imaging, and RNA-Seq, that ES cell-derived macrophages responded to S. Typhimurium, in a comparable manner to mouse bone marrow derived macrophages. We constructed a homozygous mutant mouse ES cell line in the Traf2 gene that is known to play a role in tumour necrosis factor-α signalling but has not been studied for its role in infections or response to Toll-like receptor agonists. Interestingly, traf2-deficient macrophages produced reduced levels of inflammatory cytokines in response to lipopolysaccharide (LPS) or flagellin stimulation and exhibited increased susceptibility to S. Typhimurium infection.

Histone modifications silence the GATA transcription factor genes in ovarian cancer.

Altered expression of GATA factors was found and proposed as the underlying mechanism for dedifferentiation in ovarian carcinogenesis. In particular, GATA6 is lost or excluded from the nucleus in 85% of ovarian tumors and GATA4 expression is absent in majority of ovarian cancer cell lines. Here, we evaluated their DNA and histone epigenetic modifications in five ovarian epithelial and carcinoma cell lines (human 'immortalized' ovarian surface epithelium (HIO)-117, HIO-114, A2780, SKOV3 and ES2). GATA4 and GATA6 gene silencing was found to correlate with hypoacetylation of histones H3 and H4 and loss of histone H3/lysine K4 tri-methylation at their promoters in all lines. Conversely, histone H3/lysine K9 di-methylation and HP1gamma association were not observed, excluding reorganization of GATA genes into heterochromatic structures. The histone deacetylase inhibitor trichostatin A, but not the DNA methylation inhibitor 5'-aza-2'-deoxycytidine, re-established the expression of GATA4 and/or GATA6 in A2780 and HIO-114 cells, correlating with increased histone H3 and H4 acetylation, histone H3 lysine K4 methylation and DNase I sensitivity at the promoters. Therefore, altered histone modification of the promoter loci is one mechanism responsible for the silencing of GATA transcription factors and the subsequent loss of a target gene, the tumor suppressor Disabled-2, in ovarian carcinogenesis.

The role of provocative discography in minimally invasive selective endoscopic discectomy.

A discogenic etiology is being widely accepted as a primary source of chronic low-back pain. Even though increasing information is available on the pathophysiology of the degenerated disc, the present MR techniques are still not capable of pin-pointing the source of this pain. In other words, with a non-invasive MR1 study we can still not define which disc is painful and what are the characteristics of discogenic pain from an imaging perspective. Discography remains therefore the only functional test that can delimit both morphologically and by provocation which discs are involved in the patents clinical picture. In combination with endoscopic procedures it helps tailor treatments. We consider that this diagnostic tool should be used routinely and performed by the surgeon himself as part of a screening-therapy protocol in spine surgery. We present our considerations regarding this diagnostic-screening method based on our clinical and surgical experience in patients who have been evaluated and have undergone selective endoscopic disscetomy after the screening.

Detection of simple mutations and polymorphisms in large genomic regions.

We have developed a novel technology that makes it possible to detect simple nucleotide polymorphisms directly within a sample of total genomic DNA. It allows, in a single Southern blot experiment, the determination of sequence identity of genomic regions with a combined length of hundreds of kilobases. This technology does not require PCR amplification of the target DNA regions, but exploits preparative size-fractionation of restriction-digested genomic DNA and a newly discovered property of the mismatch-specific endonuclease CEL I to cleave heteroduplex DNA with a very high specificity and sensitivity. We have used this technique to detect various simple mutations directly in the genomic DNA of isogenic pairs of recombinant Pseudomonas aeruginosa, Escherichia coli and Salmonella isolates. Also, by using a cosmid DNA library and genomic fractions as hybridization probes, we have compared total genomic DNA of two clinical P.aeruginosa clones isolated from the same patient, but exhibiting divergent phenotypes. The mutation scan correctly detected a GA insertion in the quorum-sensing regulator gene rhlR and, in addition, identified a novel intragenomic polymorphism in rrn operons, indicating very high stability of the bacterial genomes under natural non-mutator conditions.

The evolution of percutaneous spinal endoscopy and discectomy: state of the art.

OBJECTIVE: The author relates his 7 years' experience with endoscopic spine surgery for lumbar disc herniations and conditions previously treated only with more invasive methods. MATERIALS AND METHODS: Five hundred (500) patients were treated with the Yeung endoscopic spine system, which features an endoscope with a 2.8 mm operating channel. The protocol included preoperative or intraoperative discography in all cases. Adjuvant therapies were employed in various clinical conditions when dictated by the visualized spinal pathology--KTP laser (Laserscope, San Jose, CA) in 100 patients, radiofrequency by electrothermal probe in 400 patients, chymopapain in 50 cases, and intraoperative steroids in 100 cases. A newer slotted tube system allowed for foraminoplasty and removal of osteophytes or extruded fragments. RESULTS: Good-to-excellent results were recorded in 432 of the 500 patients (86.4%). Separate analysis was made of the first 100 cases when the KTP laser was in use. CONCLUSIONS: The 2.8 mm operating channel scope produced clear visualization of annular tears, disc fragments, foraminal osteophytes, and the epidural space. Monitoring of the microinstruments in the disc space and spinal canal was readily accomplished. The quality of the imaging provided by discography improved the definition of the disc pathology. Adjuvant use of lasers, radiofrequency, chymopapain, and intradiscal steroids and the newer slotted tube system, have contributed to the advances in minimally invasive technique for endoscopic discectomy.

Investigation of the substrate spectrum of the human mismatch-specific DNA N-glycosylase MED1 (MBD4): fundamental role of the catalytic domain.

The human DNA repair protein MED1 (also known as MBD4) was isolated as an interactor of the mismatch repair protein MLH1 in a yeast two-hybrid screening. MED1 has a tripartite structure with an N-terminal 5-methylcytosine binding domain (MBD), a central region, and a C-terminal catalytic domain with homology to bacterial DNA damage-specific glycosylases/lyases. Indeed, MED1 acts as a mismatch-specific DNA N-glycosylase active on thymine, uracil, and 5-fluorouracil paired with guanine. The glycosylase activity of MED1 preferentially targets G:T mismatches in the context of CpG sites; this indicates that MED1 is involved in the repair of deaminated 5-methylcytosine. Interestingly, frameshift mutations of the MED1 gene have been reported in human colorectal, endometrial, and pancreatic cancers. For its putative role in maintaining genomic fidelity at CpG sites, it is important to characterize the biochemical properties and the substrate spectrum of MED1. Here we show that MED1 works under a wide range of temperature and pH, and has a limited optimum range of ionic strength. MED1 has a weak glycosylase activity on the mutagenic adduct 3,N(4)-ethenocytosine, a metabolite of vinyl chloride and ethyl carbamate. The differences in glycosylase activity on G:U and G:T substrates are not related to differences in substrate binding and likely result from intrinsic differences in the chemical step. Finally, the isolated catalytic domain of MED1 retains the preference for G:T and G:U substrates in the context of methylated or unmethylated CpG sites. This suggests that the catalytic domain is fundamental, and the 5-methylcytosine binding domain dispensable, in determining the substrate spectrum of MED1.

Biphasic kinetics of the human DNA repair protein MED1 (MBD4), a mismatch-specific DNA N-glycosylase.

The human protein MED1 (also known as MBD4) was previously isolated in a two-hybrid screening using the mismatch repair protein MLH1 as a bait, and shown to have homology to bacterial base excision repair DNA N-glycosylases/lyases. To define the mechanisms of action of MED1, we implemented a sensitive glycosylase assay amenable to kinetic analysis. We show that MED1 functions as a mismatch-specific DNA N-glycosylase active on thymine, uracil, and 5-fluorouracil when these bases are opposite to guanine. MED1 lacks uracil glycosylase activity on single-strand DNA and abasic site lyase activity. The glycosylase activity of MED1 prefers substrates containing a G:T mismatch within methylated or unmethylated CpG sites; since G:T mismatches can originate via deamination of 5-methylcytosine to thymine, MED1 may act as a caretaker of genomic fidelity at CpG sites. A kinetic analysis revealed that MED1 displays a fast first cleavage reaction followed by slower subsequent reactions, resulting in biphasic time course; this is due to the tight binding of MED1 to the abasic site reaction product rather than a consequence of enzyme inactivation. Comparison of kinetic profiles revealed that the MED1 5-methylcytosine binding domain and methylation of the mismatched CpG site are not required for efficient catalysis.

Porphobilinogen synthase from pea: expression from an artificial gene, kinetic characterization, and novel implications for subunit interactions.

Porphobilinogen synthase (PBGS) is present in all organisms that synthesize tetrapyrroles such as heme, chlorophyll, and vitamin B(12). The homooctameric metalloenzyme catalyzes the condensation of two 5-aminolevulinic acid molecules to form the tetrapyrrole precursor porphobilinogen. An artificial gene encoding PBGS of pea (Pisum sativum L.) was designed to overcome previous problems during bacterial expression caused by suboptimal codon usage and was constructed by recursive polymerase chain reaction from synthetic oligonucleotides. The recombinant 330 residue enzyme without a putative chloroplast transit peptide was expressed in Escherichia coli and purified in 100-mg quantities. The specific activity is protein concentration dependent, which indicates that a maximally active octamer can dissociate into less active smaller units. The enzyme is most active at slightly alkaline pH; it shows two pK(a) values of 7.4 and 9.7. Atomic absorption spectroscopy shows maximal binding of three Mg(II) per subunit; kinetic data support two functionally distinct types of Mg(II) and the third appears to be nonphysiologic and inhibitory. Analysis of the protein concentration dependence of the specific activity suggests that the minimal functional unit is a tetramer. A model of octameric pea PBGS was built to predict the location of intermolecular disulfide linkages that were revealed by nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. As verified by site-specific mutagenesis, disulfide linkages can form between four cysteines per octamer, each located five amino acids from the C-terminus. These data are consistent with the protein undergoing conformational changes and the idea that whole-body motion can occur between subunits.

Purification, cloning, and characterization of the CEL I nuclease.

CEL I, isolated from celery, is the first eukaryotic nuclease known that cleaves DNA with high specificity at sites of base-substitution mismatch and DNA distortion. The enzyme requires Mg(2+) and Zn(2+) for activity, with a pH optimum at neutral pH. We have purified CEL I 33 000-fold to apparent homogeneity. A key improvement is the use of alpha-methyl-mannoside in the purification buffers to overcome the aggregation of glycoproteins with endogenous lectins. The SDS gel electrophoresis band for the homogeneous CEL I, with and without the removal of its carbohydrate moieties, was extracted, renatured, and shown to have mismatch cutting specificity. After determination of the amino acid sequence of 28% of the CEL I polypeptide, we cloned the CEL I cDNA. Potential orthologs are nucleases putatively encoded by the genes BFN1 of Arabidopsis, ZEN1 of Zinnia, and DSA6 of daylily. Homologies of CEL I with S1 and P1 nucleases are much lower. We propose that CEL I exemplifies a new family of neutral pH optimum, magnesium-stimulated, mismatch duplex-recognizing nucleases, within the S1 superfamily.

An artificial gene for human porphobilinogen synthase allows comparison of an allelic variation implicated in susceptibility to lead poisoning.

Porphobilinogen synthase (PBGS) is an ancient enzyme essential to tetrapyrrole biosynthesis (e.g. heme, chlorophyll, and vitamin B(12)). Two common alleles encoding human PBGS, K59 and N59, have been correlated with differential susceptibility of humans to lead poisoning. However, a model for human PBGS based on homologous crystal structures shows the location of the allelic variation to be distant from the active site with its two Zn(II). Previous microbial expression systems for human PBGS have resulted in a poor yield. Here, an artificial gene encoding human PBGS was constructed by recursive polymerase chain reaction from synthetic oligonucleotides to rectify this problem. The artificial gene was made to resemble the highly expressed homologous Escherichia coli hemB gene and to remove rare codons that can confound heterologous protein expression in E. coli. We have expressed and purified recombinant human PBGS variants K59 and N59 in 100-mg quantities. Both human PBGS proteins purified with eight Zn(II)/octamer; Zn(II) binding was shown to be pH-dependent; and Pb(II) could displace some of the Zn(II). However, there was no differential displacement of Zn(II) by Pb(II) between K59 and N59, and simple Pb(II) inhibition studies revealed no allelic difference.

MED1, a novel human methyl-CpG-binding endonuclease, interacts with DNA mismatch repair protein MLH1.

The DNA mismatch repair (MMR) is a specialized system, highly conserved throughout evolution, involved in the maintenance of genomic integrity. To identify novel human genes that may function in MMR, we employed the yeast interaction trap. Using the MMR protein MLH1 as bait, we cloned MED1. The MED1 protein forms a complex with MLH1, binds to methyl-CpG-containing DNA, has homology to bacterial DNA repair glycosylases/lyases, and displays endonuclease activity. Transfection of a MED1 mutant lacking the methyl-CpG-binding domain (MBD) is associated with microsatellite instability (MSI). These findings suggest that MED1 is a novel human DNA repair protein that may be involved in MMR and, as such, may be a candidate eukaryotic homologue of the bacterial MMR endonuclease, MutH. In addition, these results suggest that cytosine methylation may play a role in human DNA repair.

Incision at nucleotide insertions/deletions and base pair mismatches by the SP nuclease of spinach.

Spinach leaves contain a highly active nuclease called SP. The purified enzyme incises single-stranded DNA, RNA, and double-stranded DNA that has been destabilized by A-T-rich regions and DNA lesions [Strickland et al. (1991) Biochemistry 30, 9749-9756]. This broad range of activity has suggested that SP may be similar to a family of nucleases represented by S1, P1, and the mung bean nuclease. However, unlike these single-stranded nucleases that require acidic pH and low ionic strength conditions, SP has a neutral pH optimum and is active over a wide range of salt concentrations. We have extended these findings and showed that an outstanding substrate for SP is a mismatched DNA duplex. For base-substitution mismatches, SP incises at all mismatches except those containing a guanine residue. SP also cuts at insertion/deletions of one or more nucleotides. Where the extrahelical DNA loop contains one nucleotide, the preference of extrahelical nucleotide is A >> T approximately C but undetectable at G. The inability of SP to cut at guanine residues and the favoring of A-T-rich regions distinguish SP from the CEL I family of neutral pH mismatch endonucleases recently discovered in celery and other plants [Oleykowski et al. (1998) Nucleic Acids Res. 26, 4597-4602]. SP, like CEL I, does not turn over after incision at a mismatched site in vitro. Similar to CEL I, the presence of a DNA polymerase or a DNA ligase allows SP to turn over and stimulate its activity in vitro by about 20-fold. The possibility that the SP nuclease may be a natural variant of the CEL I family of mismatch endonucleases is discussed.

Minimally Invasive Disc Surgery with the Yeung Endoscopic Spine System (YESS).

In the 1930s, Mixter and Barr reported their results with laminectomy and discectomy, setting forth the concept that radicular pain is associated with disc herniation. Since then, investigators have attempted to findalternatives to laminectomy and discectomy to enhance operative efficacy and decrease postoperative complications such as destabilizing and scarring the spinal canal. The trend has been toward minimizing the invasiveness of procedures that currently includes microdiscectomy and percutaneous procedures.

Mutation detection using a novel plant endonuclease.

We have discovered a useful new reagent for mutation detection, a novel nuclease CEL I from celery. It is specific for DNA distortions and mismatches from pH 6 to 9. Incision is on the 3'-side of the mismatch site in one of the two DNA strands in a heteroduplex. CEL I-like nucleases are found in many plants. We report here that a simple method of enzyme mutation detection using CEL I can efficiently identify mutations and polymorphisms. To illustrate the efficacy of this approach, the exons of the BRCA1 gene were amplified by PCR using primers 5'-labeled with fluorescent dyes of two colors. The PCR products were annealed to form heteroduplexes and subjected to CEL I incision. In GeneScan analyses with a PE Applied Biosystems automated DNA sequencer, two independent incision events, one in each strand, produce truncated fragments of two colors that complement each other to confirm the position of the mismatch. CEL I can detect 100% of the sequence variants present, including deletions, insertions and missense alterations. Our results indicate that CEL I mutation detection is a highly sensitive method for detecting both polymorphisms and disease-causing mutations in DNA fragments as long as 1120 bp in length.

Binding of the Escherichia coli UvrAB proteins to the DNA mono- and diadducts of cis-[N-2-amino-N-2-methylamino-2,2,1-bicycloheptane]dichloroplatinum(II ) and cisplatin. Analysis of the factors controlling recognition and proof of monoadduct-mediated UvrB-DNA cross-linking.

The interactions of the Escherichia coli endonuclease UvrAB proteins with the DNA mono- and diadducts of both the cis-racemic exo-[N-2-amino-N-2-methylamino-2,2,1-bicycloheptane]dichloroplatin um(II) (complex 1) and cisplatin (cis-diamminedichloroplatinum(II) (cis-DDP)), have been studied. Complex 1 reacts faster with DNA than cis-DDP and gives monoadducts with a longer lifetime (8 h 20 min chelation t 1/2 compared with 2 h 40 min for cis-DDP). Using pSP65 plasmid [3H]DNA, the filter binding assay was associated with the analysis of the nucleoprotein complexes to characterize the UvrAB recognition of the platinum adducts and to demonstrate the occurrence of platinum-mediated DNA-protein cross-linking. First, it is shown that the UvrAB proteins recognize the complex 1 mono- and diadducts with a higher affinity than those of cis-DDP. Fifteen times more cis-DDP adducts per plasmid are required than complex 1 adducts, to lead to similar UvrAB binding. However, the UvrAB proteins recognize monoadducts and diadducts of each complex with a similar affinity. Second, it is shown that UvrB is the protein involved in the nucleo-protein complexes formed from mono- and diadducts of complex 1 and cis-DDP. This protein is also partly cross-linked to DNA with a similar efficiency by monoadducts derived from complex 1 and cis-DDP. However, as UvrB has a greater affinity for the DNA adducts of complex 1 than for those of cis-DDP, more UvrB-platinum-DNA cross-links are formed with complex 1 than with cis-DDP. This study, using a bacterial repair system as a model, points to a possible strategy for making new cytotoxic platinum complexes for mammalian cells.

The reactivity of 4,5',8-trimethylpsoralen with oligonucleotides containing AT sites.

Pyrimidine bases of duplex DNA, of appropriate sequence context, are photoreactive toward 4,5',8-trimethylpsoralen in the presence of long-wavelength UV light. It is generally believed that a 5'-AT site is less photoreactive with psoralen than a 5'-TA site. We have compared the reactivities of these two sites using oligonucleotide duplexes of different sequence context and found that 5'-TA and 5'-AT sites are equally reactive in certain sequences. The presence of alternating pyrimidine and purine (5'-PyATPu-3') bases in oligonucleotide duplexes optimizes the reactivity of 4,5',8-trimethylpsoralen in the 5'-AT sites.

Role of platinum-DNA adduct formation and removal in cisplatin resistance in human ovarian cancer cell lines.

A series of cisplatin-resistant cell lines were used to examine the formation and removal of platinum-DNA adducts from the overall genome and the formation and removal of cisplatin-interstrand cross-links from specific genomic regions. Cisplatin accumulation and DNA platination levels, which correlated linearly, were similar in three of the resistant cell lines despite differences in their primary cisplatin resistance. Increased platinum removal from total genomic DNA was found to be associated with increased resistance. Interstrand cross-link levels were found to be 2- to 4-fold lower in the 28S ribosomal RNA gene and a non-coding genomic region of the resistant cell lines as compared with the parental A2780 cell line. In addition, 1.2- to 2.7-fold more cross-links were formed in the non-coding region than in the ribosomal RNA gene in all of the cell lines. Interstrand cross-links were removed more rapidly from both regions of the highly cisplatin-resistant C80 and C200 cells and from the ribosomal RNA gene only in the cell lines of lower resistance. The results support a role for DNA repair and alterations in interstrand cross-link formation in cisplatin resistance and provide evidence for heterogeneous interstrand cross-link formation in the genome.

Sequence-specific interactions of UvrABC endonuclease with psoralen interstrand cross-links.

The nature of the Uvr protein-DNA complexes formed on psoralen-DNA interstrand cross-links was analyzed by DNase I footprinting and correlated with the incision efficiency of the UvrABC endonuclease on the cross-links of different DNA sequences. Our results indicate that the repair specificity is dependent on the DNA sequence and the psoralen orientation in the cross-link. On the strand that will be cut, a 30-nucleotide long UvrAB footprint with a DNase I hypersensitive site at the 11th nucleotide 5' to the lesion was observed and subsequently rearranged to a 22-nucleotide long UvrB-lesion footprint. On the strand that will not be cut, the UvrAB-lesion footprint had no 5' DNase I hypersensitive site and did not form the UvrB-lesion footprint. Although UvrABC incision requires the formation of UvrB-lesion complex on the strand which will be cut, the affinities of these complexes do not correlate with the incision efficiencies, suggesting that the overall reaction can be driven forward by a favorable next step such as UvrC incision. A study of the time-dependent interconversion of UvrAB-lesion complex to UvrB-lesion complex on a cross-link revealed a secondary recognition of the UvrB-lesion complex by UvrA2(B) proteins in vitro.