Giant Shapiro resonances in a flux-driven necklace of Josephson junctions.
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Journal Article↗
Biomedical subjects
G Chu
Publications and source records attributed to G Chu.
Purification and characterization of a human protein that binds to damaged DNA.
Xeroderma pigmentosum (XP) is an inherited disease characterized by defective repair of DNA damaged by ultraviolet (UV) radiation or agents that produce bulky DNA adducts. Human cells contain a factor that is deficient in a subset of patients from XP complementation group E and binds to DNA damaged by UV, cisplatin, or denaturation. This factor, XPE binding factor (XPE-BF), was purified to near homogeneity. The denatured protein migrated as a 125-kDa polypeptide on SDS-PAGE, and the native protein migrated primarily as a monomer on gel filtration and glycerol gradient sedimentation. Sedimentation revealed major peak in binding activity at 6.8 S, corresponding to the monomeric form, and a minor peak at 14.5 S, suggesting a homodimeric form. Binding activity was dependent on unmodified cysteine residues, stimulated by magnesium, and inhibited by zinc. Binding to UV-damaged nucleotides was 500,000-fold greater than for intact nucleotides, explaining how a molecule with an abundance of only 1-2 molecules per megabase can survey the genome for damaged DNA. Binding required a minimal DNA substrate of between 16 and 26 bp, as determined by a novel "shoe size" assay. Consistent with its previously noted versatility, XPE-BF bound to some cyclobutane pyrimidine dimers and at least one other UV-induced lesion. However, it may not bind to a subset of cyclobutane dimers, likely including the thymine dimer. These findings may explain the relatively mild phenotype of XP group E and suggest the existence of at least one other binding protein involved in the XP repair pathway.
Base Sequence↗
Bag model for DNA migration during pulsed-field electrophoresis.
A model for pulsed-field electrophoresis was developed by picturing large DNA as a deformable "bag" that (i) moves with limiting mobility in a continuous electric field, (ii) adopts an orientation aligned with the field direction, and (iii) reorients after a change in field direction in a size-dependent manner. The model correctly predicted the resolution of large DNA in a pulsed field including the surprising phenomena of mobility inversion, lateral band spreading, and improved resolution for obtuse angles. A simple parametrization agreed with observations of two completely different aspects of DNA behavior: bulk mobility as measured during gel electrophoresis and molecular reorientation as measured by linear dichroism. The model also provides quantitative guidelines for setting experimental parameters in pulsed-field electrophoresis experiments.
DNA↗
Enhanced detection of the t(14;18) translocation in malignant lymphoma using pulsed-field gel electrophoresis.
The t(14;18) chromosomal translocation that results in the juxtaposition of the bcl-2 proto-oncogene with the heavy chain JH locus is a common cytogenetic abnormality in human lymphoma. In particular, it is seen in about 85% of follicular lymphoma (FL) and up to one-third of diffuse lymphomas (DL). The chromosome 18 breakpoints have been shown to cluster into two regions. The major breakpoint region (mbr) within the 3' untranslated region of the bcl-2 proto-oncogene accounts for approximately 60% of the cases and the minor cluster region (mcr) 30 kb 3' of bcl-2 accounts for approximately 25% of the breakpoints. Because of variability in the position of the breakpoint, detection of the t(14;18) by Southern blot analysis provides an important clonal marker for the tumor. However, conventional electrophoresis (CE) fails to detect the translocation in 15% to 25% of cases. We have applied pulsed-field gel electrophoresis (PFGE) to the detection of the t(14;18) in a series of lymphoma prospectively analyzed by CE, polymerase chain reaction (PCR), and cytogenetic analysis. PFGE readily detected t(14;18) rearrangements as indicated by comigration of bands detected with probes for the mbr region (chromosome 18) and the JH locus (chromosome 14). In a series of 40 patients with FL, this method proved to be the most comprehensive for detection of the translocation compared with standard methods; in fact, in one case only PFGE was able to detect the chromosomal rearrangement. Ten percent of the FL cases were negative by all methods tested. In a separate analysis of matched tissue specimens from cases of tumor progression of FL to diffuse lymphoma, PFGE detected a common t(14;18) rearrangement confirming a clonal origin in seven of seven cases, whereas CE detected a rearrangement in only three of seven cases. Overall, PFGE was able to detect a translocation in 8 of 12 cases that were negative by CE and four of eight negative by cytogenetic analysis. In conclusion, PFGE analysis is more comprehensive than CE, PCR, and cytogenetic analysis for the detection of the t(14;18) breakpoint in tissue biopsies of malignant lymphoma.
Chromosome Mapping↗
Separation of large DNA by a variable-angle contour-clamped homogeneous electric field apparatus.
A device for separating large DNA molecules by pulsed field electrophoresis is described. Based on the principles of contour-clamped homogeneous electric fields (CHEF), it uses feedback to clamp voltages in a square electrode array, which is compact and inexpensive to construct, adaptable to computer control, and reorients the electric field by arbitrary angles. To illustrate its capabilities, pulsed fields with reorientation angles ranging from 90 to 140 degrees were used to separate DNAs of 4.7 and 5.7 megabases by up to four band-widths in 20 h. The combination of accessible technology and complete control of the electric field should facilitate the search for ways to resolve even larger DNA.
Buffers↗
Bone-inducing implants in head and neck surgery: an experimental study.
The use of autologous bone for head and neck reconstruction requires a separate harvesting procedure which provides limited quantities of bone that may become infected or undergo resorption after being implanted. In this study, a collagen/ceramic carrier containing osteoinductive factor extract (OFE) was used in a rabbit facial augmentation model. Bone-inducing activity of these implants were evaluated in subcutaneous, intramuscular, and subperiosteal sites. Implants with (test) and without OFE (control) were placed on opposite sides of the face in 40 rabbits, and were harvested at 21 days. Bone formation was evaluated by implant alkaline phosphatase determinations and histomorphometry. Osteoblastic activity, bone formation, and preservation of facial augmentation were noted in the OFE implants, showing maximal bone formation when implanted subperiosteally. Control (no OFE) and demineralized bone implants showed no bone formation. Before these implants can be used clinically, novel bone-inducing factors must be manufactured by recombinant deoxyribonucleic acid (DNA) methodology to verify activity of the homogeneous molecule which would be free of other proteins or infectious agents.
Alkaline Phosphatase↗
Transnasal butorphanol: a new method for pain relief in post-cesarean section pain.
This study was undertaken to evaluate the efficacy and the safety of transnasal butorphanol (TNB) compared to intravenous butorphanol (IVB) in 186 patients experiencing moderate to severe post-cesarean section pain. Patients were randomly assigned to five groups in a double-blind fashion: Group I (n = 37) received 2 mg IVB, Group II (n = 38) 2 mg TNB, Group III (n = 36) 1 mg TNB followed by a repeat dose of 1 mg TNB at 60 min, Group IV (n = 38) 0.5 mg TNB followed by a repeat dose of 0.5 mg at 60 min, and Group V (n = 37) received placebo. All administrations were double dummy. Pain intensity and relief were noted and the incidence of side effects was recorded. Remedication with the same study drug was allowed up to 72 h. Onset of analgesia was more rapid in the 2 mg IV group compared to the three TN groups: 5 min vs 15 min, respectively. However, the 2 mg and the 1-1 mg TN groups had a longer duration of analgesia, approximately 4.5 h, compared to 3.0 h for the 2 mg IV group (P less than 0.05). Somnolence was dose related and was the most frequent side effect, and was less frequent when the TN dose was divided into 2 doses administered 1 h apart. Multiple doses of TNB and IVB were safe and clinically acceptable up to 3 days at all doses studied. There were no incidences of nasal mucosa irritation, or cardiovascular or respiratory depression. It is concluded that transnasal butorphanol represents a safe and effective alternative to injectable butorphanol for post-cesarean section pain and offers a better and longer duration of analgesia compared to IV butorphanol. The optimum dose seems to be 2 mg TN butorphanol and it is tolerated better when divided into 1 mg increments, given 1 h apart.
Administration, Intranasal↗
Pulsed-field electrophoresis of megabase-sized DNA.
Success in constructing a physical map of the human genome will depend on two capabilities: rapid resolution of very large DNA and identification of migration anomalies. To address these issues, a systematic exploration of pulsed-field electrophoresis conditions for separating multimegabase-sized DNA was undertaken. Conditions were found for first liberating and then separating DNA up to 6 megabases at higher field strengths and more rapidly than previously reported. In addition, some conditions for transversely pulsed fields produced mobility inversion, in which increased size was accompanied by faster rather than slower migration. Importantly, anomalous migration could be identified by the presence of lateral band spreading, in which the DNA band remained sharply defined but spread laterally while moving down the gel. These results have implications for both practical applications and theoretical models of pulsed-field electrophoresis.
Blotting, Southern↗
Cisplatin-resistant cells express increased levels of a factor that recognizes damaged DNA.
Cancer treatment with the drug cisplatin is often thwarted by the emergence of drug-resistant cells. To study this phenomenon, we identified two independent cellular factors that recognize cisplatin-damaged DNA. One of the two factors, designated XPE binding factor, is deficient in complementation group E of xeroderma pigmentosum, an inherited disease characterized by defective repair of DNA damaged by ultraviolet radiation, cisplatin, and other agents. Human tumor cell lines selected for resistance to cisplatin showed more efficient DNA repair and increased expression of XPE binding factor. These results suggest that XPE binding factor may be responsible, at least in part, for the development of cisplatin resistance in human tumors and that the mechanism may be increased DNA repair.
Cell Line↗
How cells recognize damaged DNA: clues from xeroderma pigmentosum and yeast.
Xeroderma pigmentosum (XP) is characterized by the defective excision repair of DNA damaged by many agents, including ultraviolet radiation (UV) and cisplatin. We have identified a factor in human cells that recognizes multiple forms of DNA damage and is absent in XP complementation group E. Denoted XPE binding factor, it is expressed at five-fold higher levels in tumor cell lines resistant to the antitumor drug cisplatin. Finally, although it does not have photoreactivating activity, XPE binding factor shares multiple binding characteristics with yeast photolyase, suggesting that it is the human homolog of photolyase.
Cells, Cultured↗
Evidence that xeroderma pigmentosum cells from complementation group E are deficient in a homolog of yeast photolyase.
Xeroderma pigmentosum (XP) patients are deficient in the excision repair of damaged DNA. Recognition of the DNA lesion appears to involve a nuclear factor that is defective in complementation group E (XPE binding factor). We have now identified a factor in the yeast Saccharomyces cerevisiae that shares many properties with XPE binding factor, including cellular location, abundance, magnesium dependence, and relative affinities for multiple forms of damaged DNA. Yeast binding activity is dependent on photolyase, which catalyzes the photoreactivation of pyrimidine dimers. These results suggest that yeast photolyase may also function as an auxiliary protein in excision repair. Furthermore, XPE binding factor appears to be the human homolog of yeast photolyase.
Amino Acid Sequence↗
Xeroderma pigmentosum group E cells lack a nuclear factor that binds to damaged DNA.
The disease xeroderma pigmentosum is characterized by deficient repair of damaged DNA. Fusions of cells from different patients have defined nine genetic complementation groups (A through I), implying that DNA repair in humans involves multiple gene products. In this report, an extension of the gel electrophoresis binding assay was used to identify at least one nuclear factor that (i) bound to DNA damaged by ultraviolet radiation or the antitumor drug cisplatin, but (ii) was notably absent in cells from complementation group E. Therefore, the factor appears to participate in a versatile DNA repair pathway at the stage of binding and recognition.
Cell Line↗
Electroporation for the efficient transfection of mammalian cells with DNA.
A simple and reproducible procedure for the introduction of DNA into mammalian cells by electroporation is described. The parameters involving the cells, the DNA, and the electric field are investigated. The procedure has been applied to a broad range of animal cells. It is capable of transforming more than 1% of the viable cells to the stable expression of a selectable marker.
Acetyltransferases↗
DNA cross-linked by cisplatin: a new probe for the DNA repair defect in xeroderma pigmentosum.
Xeroderma pigmentosum (XP) is an inherited disease characterized by the defective repair of DNA damaged by ultraviolet radiation and a number of chemicals. In this paper, plasmid DNA carrying a marker gene is cross-linked in vitro by the antitumor drug cisplatin and successfully introduced into tissue culture cells by both calcium phosphate coprecipitation and electroporation. Transient expression of the marker gene is greatly decreased in XP cells compared to wild-type. As few as seven lesions will inactivate the marker gene in XP cells. Furthermore, the biochemical defect must include an impaired capacity for repair of cisplatin-DNA intrastrand cross-links. Since the host cell itself is not exposed to chemical modification, a cisplatin cross-linked plasmid shuttle vector can be used as a specific probe for the DNA repair capacity of cultured cells. Paradoxically, when cisplatin cross-linked plasmid carrying the selectable marker neo is introduced into cells, there is an increase in the number of stable neo+ transformants in both XP and wild-type cells. Thus, cisplatin damage appears to stimulate the integration of transfected DNA into the host chromosome by a mechanism that is independent of the defective repair pathway in XP.
Cell Line↗
Separation of large DNA molecules by contour-clamped homogeneous electric fields.
Electric fields can be manipulated by a method in which multiple electrodes are arranged along a closed contour and clamped to predetermined electric potentials. This method may be applied to a broad range of problems in the separation of macromolecules by gel electrophoresis. DNA molecules as large as 2 megabases can be well separated with a contour-clamped homogeneous electric field alternating between two orientations 120 degrees apart. The pattern of separation is independent of position in the gel, which is an advantage over previous methods. DNA less than 50 kilobases can be separated without distortion even at high voltage with a nonalternating contour-clamped homogeneous field. Decreased band broadening in DNA less than 200 bases can be achieved with a contour-clamped inhomogeneous field.
DNA↗
Rapid assay for detection of Escherichia coli xanthine-guanine phosphoribosyltransferase activity in transduced cells.
Cultured mammalian cells transduced with the Escherichia coli gene, Ecogpt, synthesize the bacterial enzyme xanthine-guanine phosphoribosyl transferase (XGPT) (1). This paper describes a method for measuring XGPT activity in crude cell extracts by following the conversion of 14C-xanthine (X) to 14C-xanthine monophosphate (XMP) and 14C-xanthosine (XR) by thin layer chromatography. The method is rapid, easy to use, sensitive and linear over a wide range of XGPT activity and has been useful for detecting XGPT in cells that were transiently transfected or stably transformed with Ecogpt. During our studies, we have found that a human cell line (XP20S) converts xanthine to XMP. This activity is probably catalyzed by a variant hypoxanthine-guanine phosphoribosyltransferase (HGPT) since the low activity is readily inhibited by hypoxanthine. A low level of conversion of X to XMP may explain why some cell lines are not killed in a medium containing mycophenolic acid and X.
Animals↗
RAD3 gene of Saccharomyces cerevisiae: nucleotide sequence of wild-type and mutant alleles, transcript mapping, and aspects of gene regulation.
We determined the complete nucleotide sequence of the RAD3 gene of Saccharomyces cerevisiae. The coding region of the gene contained 2,334 base pairs that could encode a protein with a calculated molecular weight of 89,796. Analysis of RAD3 mRNA by Northern blots and by S1 nuclease mapping indicated that the transcript was approximately 2.5 kilobases and did not contain intervening sequences. Fusions between the RAD3 gene and the lac'Z gene of Escherichia coli were constructed and used to demonstrate that the RAD3 gene was not inducible by DNA damage caused by UV radiation or 4-nitroquinoline-1-oxide. Two UV-sensitive chromosomal mutant alleles of RAD3, rad3-1 and rad3-2, were rescued by gap repair of a centromeric plasmid, and their sequences were determined. The rad3-1 mutation changed a glutamic acid to lysine, and the rad3-2 mutation changed a glycine to arginine. Previous studies have shown that disruption of the RAD3 gene results in loss of an essential function and is associated with inviability of haploid cells. In the present experiments, plasmids carrying the rad3-1 and rad3-2 mutations were introduced into haploid cells containing a disrupted RAD3 gene. These plasmids expressed the essential function of RAD3 but not its DNA repair function. A 74-base-pair deletion at the 3' end of the RAD3 coding region or a fusion of this deletion to the E. coli lac'Z gene did not affect either function of RAD3.
Alleles↗
Surgical availability of the plantaris tendon.
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Female↗