Gain of chromosome 6p is an infrequent cause of increased PIM1 expression in B-cell non-Hodgkin's lymphomas.
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Chromosomes, Human, Pair 6↗
Biomedical subjects
D Mu
Publications and source records attributed to D Mu.
Translocation t(14;18) and gain of chromosome 18/BCL2: effects on BCL2 expression and apoptosis in B-cell non-Hodgkin's lymphomas.
Gain of chromosome 18q and translocation t(14;18) are] frequently found in B-cell non-Hodgkin's lymphomas (B-NHL). Increased BCL2 transcription and BCL2 protein expression have been suggested to be the result of the gain. We utilized FISH, PCR and array CGH to study BCL2 and chromosome 18 copy number changes and rearrangements in 93 cases of B-NHL. BCL2 protein was expressed in >75% of the tumor cells in 92% of the cases by immunohistochemistry. Gain of BCL2 was associated with a 25% increase in BCL2 expression levels (immunoblotting), whereas t(14;18) resulted in a 55% increase in BCL2 levels compared to cases without BCL2 alterations. The tumor cell (spontaneous) apoptotic fractions were similar for the cases with different BCL2 genotypes. However, the normal cell apoptotic fractions were higher for the tumors with t(14;18) compared to the tumors without BCL2 alterations, while the tumors with gain of BCL2 only showed intermediate levels. Low-level gains of parts of chromosome 18 were found in 14 of the 38 B-NHL cases with t(14;18), with a consensus region 18pter-q21.33 that did not include the BCL2 gene. The 11 cases with 18q gain only showed a consensus region encompassing 18q21.2-18q21.32 and 18q21.33, which contain PMAIP1/MALT1 and BCL2, respectively.
Apoptosis↗
DNA interstrand cross-links induce futile repair synthesis in mammalian cell extracts.
DNA interstrand cross-links are induced by many carcinogens and anticancer drugs. It was previously shown that mammalian DNA excision repair nuclease makes dual incisions 5' to the cross-linked base of a psoralen cross-link, generating a gap of 22 to 28 nucleotides adjacent to the cross-link. We wished to find the fates of the gap and the cross-link in this complex structure under conditions conducive to repair synthesis, using cell extracts from wild-type and cross-linker-sensitive mutant cell lines. We found that the extracts from both types of strains filled in the gap but were severely defective in ligating the resulting nick and incapable of removing the cross-link. The net result was a futile damage-induced DNA synthesis which converted a gap into a nick without removing the damage. In addition, in this study, we showed that the structure-specific endonuclease, the XPF-ERCC1 heterodimer, acted as a 3'-to-5' exonuclease on cross-linked DNA in the presence of RPA. Collectively, these observations shed some light on the cellular processing of DNA cross-links and reveal that cross-links induce a futile DNA synthesis cycle that may constitute a signal for specific cellular responses to cross-linked DNA.
Animals↗
The relationship between the c-jun mRNA expression and apoptosis of neurons in rat brain following perinatal ischemic-hypoxia.
OBJECTIVE: To investigate the relationship between c-jun mRNA and apoptosis of neurons following perinatal ischemic-hypoxia. METHODS: We set up a fetal rat model of perinatal ischemic-hypoxia by ligating unilateral uterine horn vessel of pregnant Wistar rats (21-day gestation). The contralateral horn vessel was not ligated and the fetuses in these uteri served as controls. Rat pups were delivered by cesarean section at the end of ischemic hypoxic insult and then the rats' brain tissues were collected in different points of time. In situ hybridization and TUNEL methods were used to detect the c-jun mRNA expression and neural cells apoptosis separately. RESULTS: The expression of c-jun mRNA in brain tissues began at 15 minutes, reached the first peak at 1-2 hours after ischemic-hypoxic insult and reduced gradually after 4 hours and the second peak at 24th hour after insult, gradually disappeared till 72nd hour. There were very low c-jun mRNA expression of hippocampus in the control group 24 h after birth. Meanwhile, we observed that the apoptosis of neuronal cells in cerebral cortex was much more than that in the control group. CONCLUSION: The increases of immediate early gene c-jun expression and the cell apoptosis could be induced by perinatal ischemic-hypoxia. The higher expression of c-jun mRNA might induce the transcription of its target gene, especially, so called "death related genes", which would be a promoter for cell apoptosis.
Animals↗
(6-4) photolyase: light-dependent repair of DNA damage.
DNA photolyase represents a phenomenal class of DNA repair enzymes in that it harvests the light energy to repair DNA lesions caused by ultraviolet light. Mother Nature evolves two types of photolyases, one specific for repairing cyclobutane pyrimidine dimers and the other for pyrimidine-(6-4)-pyrimidone photoproducts. Together, these two kinds of DNA photolesions account for the majority of ultraviolet light-induced DNA lesions. So far, the basic chemical steps of the enzyme mechanism of the two classes of photolyases appear to be very similar. Therefore, it will be very interesting to uncover the determinants of the different substrate specificity between the two photolyases. In this review, we focus on the discussion of the photolyase specific for repairing pyrimidine-(6-4)-pyrimidone photoproducts mainly because the research of the cyclobutane pyrimidine dimer photolyase has recently been reviewed quite extensively.
Animals↗
[Determination of germanium and selenium in malt powder by GFAAS].
A method for determination of germanium and selenium in malt powder has been developed by graphite furnace atomic absorption spectrometry with L'vov platform. While (Pd+Ni) and Pd were used as matrix modifiers,the ashing temperatures of Ge and Se were increased to 1400 and 1200 degrees C respectively,and the matrix inferences were removed. The characteristic masses are 31 pg Ge and 23pg Se(0.0044 A), the detection limits are 28pg Ge and 62pg Se (3 sigma), the precisions (n = 9) are 3. 7-5.6%(Ge) and 4.3-6.5% (Se) and the recoveries are among 90-105%.
Edible Grain↗
Characterization of reaction intermediates of human excision repair nuclease.
Nucleotide excision repair in humans is a complex reaction involving 14 polypeptides in six repair factors for dual incisions on either sides of a DNA lesion. To identify the reaction intermediates that form by the human excision repair nuclease, we adopted three approaches: purification of functional DNA.protein complexes, permanganate footprinting, and the employment as substrate of presumptive DNA reaction intermediates containing unwound sequences 5' to, 3' to, or encompassing the DNA lesion. The first detectable reaction intermediate was formed by substrate binding of XPA, RPA, XPC.HHR23B plus TFIIH (preincision complex 1, PIC1). In this complex the DNA was unwound on either side of the lesion by no more than 10 bases. Independent of the XPG nuclease function, the XPG protein stabilized this complex, forming a long lived preincision complex 2 (PIC2). The XPF.ERCC1 complex bound to PIC2, forming PIC3, which led to dual incisions and the release of the excised oligomer. With partially unwound DNAs, thymine cyclobutane dimer was excised at a fast rate independent of XPC.HHR23B, indicating that a major function of this protein is to stabilize the unwound DNA or to aid lesion unwinding in preincision complexes.
Base Sequence↗
Model for XPC-independent transcription-coupled repair of pyrimidine dimers in humans.
In humans, DNA lesions such as pyrimidine dimers in the template strand of genes transcribed by RNA polymerase II are repaired faster than those in the coding strand and nontranscribed regions of the genome. This phenomenon, referred to as transcription-coupled repair (i) requires active transcription, (ii) does not require the XPC gene product which is essential for general/basal repair reactions, and (iii) requires the CSA and CSB proteins. We have developed an in vitro model system that consists of purified human excision repair factors and a DNA substrate analogous to a transcription bubble terminating at a cyclobutane thymine dimer. In this system the thymine dimer was excised independent of XPC. Furthermore, the thymine dimer in the bubble-containing substrate was removed approximately 3-fold faster by the excision repair nuclease reconstituted with or without XPC, compared with the removal of thymine dimer from a base paired duplex by the entire set of excision nuclease factors. These results provide important insight into the mechanism of transcription-coupled repair in humans.
Base Sequence↗
DNA excision repair assays.
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DNA Repair↗
Initiation of DNA interstrand cross-link repair in humans: the nucleotide excision repair system makes dual incisions 5' to the cross-linked base and removes a 22- to 28-nucleotide-long damage-free strand.
Most DNA repair mechanisms rely on the redundant information inherent to the duplex to remove damaged nucleotides and replace them with normal ones, using the complementary strand as a template. Interstrand cross-links pose a unique challenge to the DNA repair machinery because both strands are damaged. To study the repair of interstrand cross-links by mammalian cells, we tested the activities of cell extracts of wild-type or excision repair-defective rodent cell lines and of purified human excision nuclease on a duplex with a site-specific cross-link. We found that in contrast to monoadducts, which are removed by dual incisions bracketing the lesion, the cross-link causes dual incisions, both 5' to the cross-link in one of the two strands. The net result is the generation of a 22- to 28-nucleotide-long gap immediately 5' to the cross-link. This gap may act as a recombinogenic signal to initiate cross-link removal.
Animals↗
Recognition and repair of compound DNA lesions (base damage and mismatch) by human mismatch repair and excision repair systems.
Nucleotide excision repair and the long-patch mismatch repair systems correct abnormal DNA structures arising from DNA damage and replication errors, respectively. DNA synthesis past a damaged base (translesion replication) often causes misincorporation at the lesion site. In addition, mismatches are hot spots for DNA damage because of increased susceptibility of unpaired bases to chemical modification. We call such a DNA lesion, that is, a base damage superimposed on a mismatch, a compound lesion. To learn about the processing of compound lesions by human cells, synthetic compound lesions containing UV photoproducts or cisplatin 1,2-d(GpG) intrastrand cross-link and mismatch were tested for binding to the human mismatch recognition complex hMutS alpha and for excision by the human excision nuclease. No functional overlap between excision repair and mismatch repair was observed. The presence of a thymine dimer or a cisplatin diadduct in the context of a G-T mismatch reduced the affinity of hMutS alpha for the mismatch. In contrast, the damaged bases in these compound lesions were excised three- to fourfold faster than simple lesions by the human excision nuclease, regardless of the presence of hMutS alpha in the reaction. These results provide a new perspective on how excision repair, a cellular defense system for maintaining genomic integrity, can fix mutations under certain circumstances.
Adaptor Proteins, Signal Transducing↗
[The changes of glutamate receptor and free Ca2+i in hypoxic-ischemic cerebral injury: experimental study].
This experiment was designed to explore the pathogenesis in hypoxic-ischemic encephalopathy (HIE). Sixteen newborn pigs were divided into two groups: (Group A) normal control and (Group B) HIE 24 hours. The glutamate receptor (Glu R) in forebrain crude synaptic membrane (SPM) and free Ca2+i in RBC were tested respectively. The results revealed that the binding sites (Bmax) of Glu R in Group B was much lower than that in Group A, but the affinity (Kd) showed no statistic difference between Group A and Group B. In addition, free Ca2+i of RBC in Group B was much higher than that in Group A. This study demonstrates that the changes of Glu R and Ca2+i are involved in the pathogenesis of hypoxic-ischemic cerebral injury in newborn animals.
Animals↗
Overproduction, purification, and characterization of the XPC subunit of the human DNA repair excision nuclease.
Xeroderma pigmentosum complementation group C gene (XPC) encodes a protein of 125 kDa which is present in a tight complex with a 58-kDa protein encoded by the human homolog of the yeast RAD23 gene, HHR23B (Masutani, C., Sugasawa, K., Yanagisawa, J., Sonoyama, T., Ui, M., Enomoto, T., Takio, K., Tanaka, K., van der Spek, P. J., Bootsma, D., Hoeijmakers, J. H. J., and Hanaoka, F.(1994) EMBO J. 13, 1831-1843). The XPC-HHR23B complex is required for excision of thymine dimers from DNA in a human excision nuclease system reconstituted from purified proteins. In order to understand the role of the XPC-HHR23B complex in excision repair, we have overexpressed each subunit alone and the heterodimer in heterologous systems, purified them, and characterized their biochemical properties. We find that both XPC and the heterodimer bind DNA with high affinity and UV-damaged DNA with slightly higher preference. Surprisingly, we find that the XPC subunit alone is sufficient for reconstitution of the human excision nuclease and that the HHR23B subunit has no detectable effect on the excision activity of the reconstituted system.
Animals↗
Repair of cisplatin--DNA adducts by the mammalian excision nuclease.
Nucleotide excision repair is one of the many cellular defense mechanisms against the toxic effects of cisplatin. An in vitro excision repair assay employing mammalian cell-free extracts was used to determine that the 1,2-d(ApG) intrastrand cross-link, a prevalent cisplatin-DNA adduct, is excised by the excinuclease from a site-specifically modified oligonucleotide 156 base pairs in length. Repair of the minor interstrand d(G)/d(G) cross-link was not detected by using this system. Proteins containing the high mobility group (HMG) domain DNA-binding motif, in particular, rat HMG1 and a murine testis-specific HMG-domain protein, specifically inhibit excision repair of the intrastrand 1,2-d(GpG) and -d(ApG) cross-links. This effect was also exhibited by a single HMG domain from HMG1. Similar inhibition of repair of a site-specific 1,2-d(GpG) intrastrand cross-link by an HMG-domain protein also occurred in a reconstituted system containing highly purified repair factors. These results indicate that HMG-domain proteins can block excision repair of the major cisplatin-DNA adducts and suggest that such an activity could contribute to the unique sensitivity of certain tumors to the drug. The reconstituted excinuclease was more efficient at excising the 1,3-d(GpTpG) intrastrand adduct than either the 1,2-d(GpG) or d(ApG) intrastrand adducts, in agreement with previous experiments using whole cell extracts [Huang, J. -C., Zamble, D. B., Reardon, J. T., Lippard, S. J., Sancar, A. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 10394-10398]. This result suggests that structural differences among the platinated DNA substrates, and not the presence of unidentified cellular factors, determine the relative excision repair rates of cisplatin-DNA intrastrand cross-links in the whole cell extracts.
Animals↗
Functional complementation of xeroderma pigmentosum complementation group E by replication protein A in an in vitro system.
Xeroderma pigmentosum (XP) is caused by a defect in nucleotide excision repair. Patients in the complementation group E (XP-E) have the mildest form of the disease and the highest level of residual repair activity. About 20% of the cell strains derived from XP-E patients lack a damaged DNA-binding protein (DDB) activity that binds to ultraviolet-induced (6-4) photoproducts with high affinity. We report here that cell-free extracts prepared from XP-E cell strains that either lacked or contained DDB activity were severely defective in excising DNA damage including (6-4) photoproducts. However, this excision activity defect was not restored by addition of purified DDB that, in fact, inhibited removal of (6-4) photoproducts by the human excision nuclease reconstituted from purified proteins. Extensive purification of correcting activity from HeLa cells revealed that the correcting activity is inseparable from the human replication/repair protein A [RPA (also known as human single stranded DNA binding protein, HSSB)]. Indeed, supplementing XP-E extracts with recombinant human RPA purified from Escherichia coli restored excision activity. However, no mutation was found in the genes encoding the three subunits of RPA in an XP-E (DDB-) cell line. It is concluded that RPA functionally complements XP-E extracts in vitro, but it is not genetically altered in XP-E patients.
Base Sequence↗
Replication protein A confers structure-specific endonuclease activities to the XPF-ERCC1 and XPG subunits of human DNA repair excision nuclease.
XPF-ERCC1 and XPG proteins are nucleases that are involved in human nucleotide excision repair. In this study, we characterized the structure-specific junction-cutting activities of both nucleases using DNA substrates containing a bubble or loop structure. We found that the junction-cutting activities of XPF-ERCC1 and XPG were greatly stimulated by human replication protein A (RPA), while heterologous single-stranded DNA-binding proteins could not substitute for human RPA. To test for specific interaction between RPA and XPF-ERCC1 as is known to occur between RPA and XPG, we employed a pull-down assay with immobilized "bubble" substrate. We found that the binding of XPF-ERCC1 complex to the bubble substrate was enhanced by RPA, suggesting a possible mechanism for RPA in the excision nuclease system, that is the targeting of the nuclease subunits to their specific sites of action. Furthermore, the RPA-promoted junction cutting by XPF-ERCC1 and XPG nucleases was observed with "loop" substrates as well, raising the possibility that XPF-ERCC1, XPG, and RPA may function in removing loop structures from DNA, independent of the other subunits of the human excinuclease.
Base Sequence↗
Reaction mechanism of human DNA repair excision nuclease.
Nucleotide excision repair consists of removal of the damaged nucleotide(s) from DNA by dual incision of the damaged strand on both sides of the lesion, followed by filling of the resulting gap and ligation. In humans, 14-16 polypeptides are required for the dual incision step. We have purified the required proteins to homogeneity and reconstituted the dual incision activity (excision nuclease) in a defined enzyme/substrate system. The system was highly efficient, removing >30% of the thymine dimers under optimal conditions. All of the six fractions that constitute the excision nuclease were required for dual incision of the thymine dimer substrate. However, when a cholesterol-substituted oligonucleotide was used as substrate, excision occurred in the absence of the XPC-HHR23B complex, reminiscent of transcription-coupled repair in the XP-C mutant cell line. Replication protein A is absolutely required for both incisions. The XPG subunit is essential to the formation of the preincision complex, but the repair complex can assemble and produce normal levels of 3'-incision in the absence of XPF-ERCC1. Kinetic experiments revealed that the 3'-incision precedes the 5'-incision. Consistent with the kinetic data, uncoupled 5'-incision was never observed in the reconstituted system. Two forms of TFIIH were used in the reconstitution reaction, one containing the CDK7-cyclin H pair and one lacking it. Both forms were equally active in excision. The excised oligomer dissociated from the gapped DNA in a nucleoprotein complex. In total, these results provide a detailed account of the reactions occurring during damage removal by human excision nuclease.
Cholesterol↗
[Dexamethasone's effect on glutamate receptor in experimental hypoxic-ischemic cerebral injury].
In order to probe into dexamethasone's effect on glutamate receptor (Glu R) in hypoxic-ischemic encephalopathy (HIE) of newborn, we established newborn pigs' model of HIE to investigate Glu R in their forebrain crude synaptic membrane and the effect of dexamethasone in different HIE times and different dosages on the changes of Glu R. This study included five groups: HIE group (no medication); dexamethasone treatment group (10 mg/kg before HIE); dexamethasone treatment group (10 mg/kg after HIE); dexamethasone treatment group (5 mg/kg before HIE); dexamethasone treatment group (5 mg/kg after HIE). The results showed that the mean receptor density (Bmax) of dexamethasone treatment group (10 mg/kg before HIE) was significantly lower than that of the other groups (P < 0.05), but there was no statistic difference in the affinity of Glu R between all the experimental groups. These findings suggested that the preventive use of dexamethasone in large dosage would reduce the binding of Glu, and could possibly protect the brain tissues from damage in HIE.
Animals↗