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PubMed1989

Compensation as a risk factor for lung cancer in smokers who switch from nonfilter to filter cigarettes.

The likelihood of increasing the number of cigarettes per day (cpd), a common method of compensation, in smokers who had switched from nonfilter to filter cigarettes was examined in newly diagnosed and histologically confirmed lung cancer cases including all cell types. There were a total of 1,242 lung cancer cases and 2,300 sex and age matched hospital controls interviewed in twenty hospitals from nine United States cities between 1969 and 1984. Lung cancer cases compensated more (as increased cpd after switching) than the controls. The mean increase in cpd for cases was about twice that of controls. Using switchers who did not increase cpd as the referent group, switchers who increased cpd showed a clearcut dose-response relationship for lung cancer with increasing levels of compensation. The odds ratio increased from 1.2 to 2.4 in males and 1.7 to 3.8 in females corresponding to increases of 1-10 to 21+ cpd after switching. Findings from this study suggest that increasing cpd after switching to filter cigarettes is an important risk factor for lung cancer that needs to be stressed in epidemiologic studies. Proponents of the idea that switching cigarettes is of some benefit, should also advocate that individuals who continue smoking should avoid compensation after switching.

Female↗
PubMed1994

A multicenter study on the hospitalization of pediatric patients on chronic peritoneal dialysis.

The Italian Registry of Pediatric Chronic Peritoneal Dialysis (CPD) carried out a special study on patient hospitalization during the years 1989-1992. Ninety-two children (mean age 8.4 +/- 4.7 years) entered the study, for a total of 1406 CPD-months. The contribution of the different causes of hospitalization for a total of 4683 hospital days was: CPD training 31%; routine controls 14%; CPD-related complications 35%; clinical complications 14%; other causes 6%. The rate of patient hospitalization that resulted was 3.33 days/CPD-month; it was higher in the first year (4.32 days/CPD-month) than in the second year (1.64 days/CPD-month) or in the third year (2.25 days/CPD-month). This difference was mainly due to the need for the training at the start of the CPD treatment. The evaluation of the hospitalization rate in different age groups showed a statistically significant difference (p < 0.05) between the group 0-2 years (5.47 days/CPD-month) and the group 3-15 years (2.78 days/CPD-month). Complications were the cause of 150 admissions to the hospital (1:9.6 CPD-months). Ninety-eight admissions were due to CPD-related complications: peritonitis (33%), problems with the catheter (19%), abdominal hernias (4%), and others (9%). Among clinical complications (52 admissions), the main cause of hospitalization was hypertension (15%), followed by infections (4%), and malnutrition (3%).

Adolescent↗
PubMed1995

Clinical experience in the treatment of infants with chronic peritoneal dialysis.

Chronic peritoneal dialysis (CPD) is the first treatment modality for most infants with end-stage renal failure; this group of patients shows peculiar clinical and technical problems. We present the data from a National Registry on 22 children starting CPD under one year of age, representing 11.6% of the total population of the Registry (189 patients). Mean weight at start of CPD was 6.1 +/- 1.8 kg and duration of dialysis was 22.1 +/- 15.5 months. During the follow-up period, 9 patients were transplanted, 1 was shifted to hemodialysis, and 4 died. Patient survival was 89.1% and 82.2% at 1 and 2 years (97.9% and 96.5% in the group of 167 older children); technique survival results were 89.1% at 1 year and 77.1% at 2 years (vs 92.5% and 85.7%, respectively). The incidence of peritonitis was 1 episode every 15.6 CPD-months (1:16.1 in the older children). Catheter-related complications occurred more frequently in infants (1:11.8 vs 1:17 episode:CPD-months), even if this difference was not statistically significant. Statural growth was on average -0.29 +/- 0.66 SD/year with a significant improvement between the first (-0.50 +/- 0.79) and the second (+0.23 +/- 0.77) year of CPD. Our data confirm that infants represent a higher risk group and that they can be treated satisfactorily with CPD while awaiting renal transplantation.

Catheters, Indwelling↗
PubMed1996

Comparison of patient hospitalization in chronic peritoneal dialysis and hemodialysis: a pediatric multicenter study.

Patient hospitalization was compared in 207 pediatric patients (age < or = 15 years at the start of dialysis) on chronic peritoneal dialysis (CPD) (127 patients) or center hemodialysis (HD) (80 patients), treated in 17 dialysis centers during the period 1989 to 1994, and followed up for at least three months. The hospitalization rate was expressed as hospital days per patient-month, and was calculated on the overall period of treatment and separately for the first and second year. Since the age at start of dialysis markedly differed between CPD (8.2 +/- 4.7 years) and HD (11.2 +/- 2.9 years) patients (with no HD patient younger than five years), results are separately presented in three patient groups: CPD patients aged < 5 years (A); CPD patients aged five to 15 years (B); HD patients (C). The duration of hospitalization was subdivided according to the following different causes: routine (monitoring of dialysis adequacy), complications of the modality, patient primary renal disease, and other causes. The results are presented in Table 1. A statistically significant difference in total days hospitalized was found between each of the two groups of CPD patients and the HD patients; the results for hospitalization for dialysis-related complications were higher in the group of younger children on CPD, while the difference between the two age-matched groups of patients on CPD and HD was not significant.

Adolescent↗
PubMed1997

Variability of peritoneal equilibration test in children on continuous peritoneal dialysis by repeated testing with solutions of different osmolarity.

To evaluate (1) differences in the peritoneal equilibration test (PET) achieved using continuous peritoneal dialysis (CPD) solutions containing different amounts of glucose and (2) intraindividual reproducibility of PETs performed twice within an interval of 8 months on CPD, we investigated 39 PETs in 13 children aged 2.4-19.0 years (median 10.6 years) on stable CPD regimens. The fill volume was 1 L/m2 body surface area. We used a standard CPD solution (Fresenius) with a 2.3% glucose content (groups 2.3a and 2.3b) two times within an interval of 1-8 months. A third test was done between the two with a CPD solution of 1.5% glucose (group 1.5). Equilibration quotients, that is, substrate concentration in dialysis fluid divided by substrate concentration in plasma (D/P), did not show any statistically significant differences between groups 1.5 and 2.3a or between groups 2.3a and 2.3b. A significant difference was seen in the decline of glucose content of dialysate between groups 1.5 and 2.3 but not between groups 2.3a and 2.3b. Ultrafiltration was higher in groups 2.3a and 2.3b compared with group 1.5. Inter- and intraindividual variability between solute transfer was small during follow-up in stable CPD patients. Different glucose contents of 1.5 and 2.3 g/dL dialysis fluid had no measurable influence on PET results of stable CPD patients. For standard PETs, reducing the glucose content of dialysis fluid to isoosmolarity is not necessary.

Adolescent↗
PubMed2006

Testing excision models for responses of mismatch-repair systems to UV photoproducts in DNA.

Mismatch-repair (MMR) systems correct DNA replication errors and respond to a variety of DNA lesions. Previous observations that MMR antagonizes UV mutagenesis, and that the mismatch-recognition protein heterodimer MSH2*MSH6 (MutSalpha) selectively binds DNA containing "mismatched" photoproducts (T[CPD]T/AG, T[6-4]T/AG) but not "matched" photoproducts (T[CPD]T/AA, T[6-4]T/AA), suggested that mismatched photoproducts would provoke MMR excision similar to mismatched bases. Excision of incorrect nucleotides inserted opposite template photoproducts might then prevent UV-induced mutation. We tested T[CPD]T/AG DNA, in a sequence context in which it is bound substantially by hMutSalpha and in three other contexts, for stimulation of 3' MMR excision in mammalian nuclear extracts. T[CPD]T/AG was inactive in HeLa extracts, or in extracts deficient in the photoproduct-binding proteins DDB or XPC* hHR23B, arguing against interference from the nucleotide excision repair pathway. Prior incubation with hMutSalpha and MLH2.PMS2 (hMutLalpha) did not increase excision relative to homoduplex controls. T[6-4]T/AG also failed to provoke excision. T/G, C/A, and T/T substrates, even though bound by hMutSalpha no better than T[CPD]T/AG substrates, efficiently provoked excision. Even a substrate containing three T[CPD]T/AG photoproducts (in different contexts) did not significantly provoke excision. Thus, MMR may suppress UV mutagenesis by non-excisive mechanisms.

Animals↗
PubMed2005

A free energy calculation study of the effect of H-->F substitution on binding affinity in ligand-antibody interactions.

Changes in binding affinity to catalytic antibody 6D9 of chloramphenicol phosphonate derivatives (CPDs) containing H or F were investigated by performing free energy calculations based on molecular dynamics simulations. We calculated the binding free energy, enthalpy, and entropy changes (DeltaDeltaG, DeltaDeltaH, and -TDeltaDeltaS) attributable to H-->F substitution by comparing results for CPDs containing a trifluoroacetylamino group (CPD-F) or an acetylamino group (CPD-H). The calculated DeltaDeltaG, DeltaDeltaH, and -TDeltaDeltaS values were -2.9, -6.3, and 3.5 kcal mol(-1) and close to experimental values observed for a series of similar ligands, chloramphenicol phosphonates with F and H (-1.4, -3.5, and 2.1 kcal mol(-1)). Therefore, CPD-F binds more strongly to 6D9 than does CPD-H. To clarify the origin of the large difference in DeltaDeltaG, we apportioned the calculated values of DeltaDeltaG and DeltaG for the associated and dissociated states into contributions from various atomic interactions. We found that the H-->F substitution increased the binding affinity mainly by decreasing the hydration free energy and not by increasing favorable interactions with the antibody. The decreased hydration free energy of the ligand was mainly due to unfavorable coulombic interactions between the trifluoroacetylamino group and solvent waters, which increased the free energy of the dissociated state (by about 3.7 kcal mol(-1)). Also, the trifluoroacetylamino group slightly increased the free energy level of the associated state (about 0.8 kcal mol(-1)) because favorable van der Waals interactions compensated for unfavorable coulombic interactions with antibody atoms. In addition, the enthalpy and entropy changes, DeltaDeltaH and -TDeltaDeltaS (computationally -6.3 and 3.5 kcal mol(-1)), originated mainly from a decrease in hydration free energy in the dissociated state. The CPD-F and CPD-H ligands had substantially different structures in the dissociated and complexed states.

Antibodies↗
PubMed2005

Hepatocyte growth factor enhances protein phosphatase Cdc25A inhibitor compound 5-induced hepatoma cell growth inhibition via Akt-mediated MAPK pathway.

We have previously shown that Compound 5 (Cpd 5), an inhibitor of protein phosphatase Cdc25A, inhibits Hep3B human hepatoma cell growth. We now show that hepatocyte growth factor (HGF), a hepatocyte growth stimulant, can strongly enhance Cpd 5-induced growth inhibition in Hep3B cells, and this enhancement in cell growth inhibition is correlated with a much stronger ERK phosphorylation when compared to cells treated with Cpd 5 or HGF separately. We found that HGF/Cpd 5-induced ERK phosphorylation and cell growth inhibition were mediated by Akt (protein kinase B) pathway, since combination HGF/Cpd 5 treatment of Hep3B cells inhibited Akt phosphorylation at Ser-473 and its kinase activity, which led to the suppression of Raf-1 phosphorylation at Ser-259. The suppression of Raf-1 Ser-259 phosphorylation caused the induction of Raf-1 kinase activity, as well as hyper-ERK phosphorylation. Transient transfection of Hep3B cells with dominant negative Akt c-DNA further enhanced both Cpd 5- and HGF/Cpd 5-induced ERK phosphorylation, while over-expression of wild-type Akt c-DNA diminished their effects. In contrast, HGF antagonized the growth inhibitory actions of Cpd 5 on normal rat hepatocytes, thus showing a selective effect on tumor cells compared to normal cells. Our data suggest that Akt kinase negatively regulates MAPK activity at the Akt-Raf level. Suppression of Akt activity by either combination HGF/Cpd 5 treatment or by dominant negative Akt c-DNA transfection antagonizes the Akt inhibitory effect on Raf-1, resulting in an enhancement of Cpd 5-induced MAPK activation and cell growth inhibition.

Animals↗
PubMed1993

Comparison of hexamethyldisilazane (HMDS), Peldri II, and critical-point drying methods for scanning electron microscopy of biological specimens.

Three different drying methods, critical-point drying (CPD), Peldri II, and hexamethyldisilazane (HMDS), were compared using representative animal (rat kidney, trachea, duodenum, lung, and red blood cells) and plant (leaves from ten species of monocotyledons and dicotyledons) specimens. All three drying methods produced identical results with animal specimens. Plant specimens showed signs of shrinkage regardless of which drying method was employed. The order of preservation quality from best to worst for leaves was CPD > Peldri II > HMDS, with the CPD method providing substantially better results in all but one case. Postfixation of leaves with osmium tetroxide resulted in poorer preservation in all instances. Peldri II caused complete extraction of leaf cuticular wax, while both both CPD and HMDS showed minimal extraction compared with samples air dried directly from acetone. These results indicate that HMDS provides a time-saving and inexpensive alternative to CPD for animal specimens. Plant specimens, particularly those containing cells with large central vacuoles, are adequately preserved only with the CPD method. In addition, postfixation with osmium should be avoided when processing plant specimens for scanning electron microscopy.

Animals↗
PubMed1978

Whole blood storage in citrate and phosphate solutions containing half-strength trisodium citrate: cellular and biochemical studies.

The efficacy of whole blood preservation in acid citrate dextrose (ACD-A) and citrate-phosphate dextrose (CPD) anticoagulants containing half-strength trisodium citrate concentrations, was determined by biochemical and cellular assessment during 28 day storage at 4 degrees--6 degrees. Erythrocyte 2,3-DPG and ATP concentrations, serum potassium, plasma haemoglobin, and blood pH values were similar between the standard and half-strength citrate counterpart in both ACD-A and CPD series, throughout the entire storage period. Erythrocyte indices (MCV, MCH, MCHC, and osmotic fragility) and the red cell, platelet, and leucocyte counts, were also similar regardless of the final citrate concentration in both ACD-A and CPD series during the 28 day storage period. Tests of coagulation (PT, KCCT, TT, and ethanol gel) and fibrinogen levels were also similar, except for a lower quantity of clottable fibrinogen at day 28 in half-strength CPD. The formation of platelet and leucocyte aggregates during storage, as measured by changes in the SFP, were similar in magnitude whether or not the half-strength citrate formulation was used in ACD-A or CPD. Phagocytic and bactericidal capacity against Staph. aureus was normal following 24-hr storage at 4 degrees--6 degrees, in both ACD-A and CPD preserved blood, regardless of the final citrate concentration. It may now be concluded from this evaluation and those of others, that a reasonable argument can be made for reducing the citrate quantity presently used during blood storage, in order to provide a safe and effective transfusion product for routine use.

Adenosine Triphosphate↗
PubMed1989

Effects of selective monoaminergic reuptake blockade on activity rhythms in developing rats.

Developing rats display prominent ultradian rhythms of locomotor activity when separated from the litter. A pharmacological analysis was undertaken to provide preliminary data on the role of monoaminergic neurotransmitter systems in the modulation or manifestation of this fundamental biological rhythm. Twenty-four hour activity profiles were monitored in 15-day-old rats, tested in darkness, after intraperitoneal treatment with desipramine (DMI), zimelidine (ZMI), or GBR-13069 (GBR), selective uptake inhibitors of norepinephrine, serotonin, and dopamine, respectively. Time series data were analyzed by low-resolution variance spectral analysis. DMI significantly diminished ultradian (greater than 1 cycle per day; cpd) rhythmicity, and enhanced the circadian rhythm. Equimolar doses of ZMI had little effect on the ultradian band (7-15 cpd), but slightly reduced the circadian peak. The effects of acute GBR administration were complex, as this agent produced prominent effects on basal activity. In a second study these agents were administered continuously over a 5-day period, using subcutaneously implanted Alzet osmotic minipumps, to avoid the confounding effects of acute administration. Continuously-infused DMI virtually eliminated characteristic ultradian rhythms in the 9-15 cpd bandwidth. ZIM diminished ultradian oscillations only in the 14-15 cpd range, and GBR-12909 had little effect on ultradian rhythms throughout the usually prominent 7-16 cpd domain. All three reuptake inhibitors increased the prominence of slow ultradian rhythms with frequencies of 3-4 cpd. Continuous reuptake blockade had no significant effects on circadian amplitude or phase, as determined by cosinor analysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1975

Mutants of Serratia marcescens lacking cyclic nucleotide phosphodiesterase activity and requiring cyclic 3',5'-AMP for the utilization of various carbohydrates.

Adenine requiring mutants of Serratia marcescens SM-6-F'lac+ have been found to grow well in minimal-glucose medium solely supplemented with cAMP. From one of these ade strains double mutants (called ade cpd) were isolated which could no longer utilize cAMP but which still grew on 5'AMP. Dialyzed cell extracts (soluble fraction) of the double mutants, assayed for cAMP phosphodiesterase, were unable to hydrolyze cAMP whereas cell extracts of the parental strains yielded 5'AMP at a rate of 1.6-2.0 mumoles min-1 mg-1 protein. The loss of the phosphodiesterase activity in S. marcescens cpd W 1181 did not cause an accumulation of large amounts of cAMP as was found for the diesterase-negative mutant AB257pc-1 of Escherichia coli. The induced synthesis of beta-galactosidase in mutant cpd W 1181 showed about the same sensitivity to transient and permanent catabolite (glucose) repression as the corresponding cpd+ strain. Starting from S. marcescens cpd W 1182 three independent double mutants (called cpd cya) were isolated which required exogenous cAMP for utilizing various carbohydrates as carbon source, for motility and for the formation of extracellular lipase and the red pigment prodigiosine. The intracellular concentration of cAMP in these mutants, grown in nutrient broth, was 40-60% of that of the parental strain which is about 4 x 10(-4) M. However, the adenylate cyclase in cell extracts of the mutants W 1237 and W 1270 was like that of the corresponding cya+ strain (about 2 x 10(-2) mumoles min-1 mg-1 protein).

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1994

Growth of children following the initiation of dialysis: a comparison of three dialysis modalities.

Maintenance dialysis usually serves as an interim treatment for children with end-stage renal disease (ESRD) until transplantation can take place. Some children, however, may require dialytic support for an extended period of time. Although dialysis improves some of the problems associated with growth failure in ESRD (acidosis, uremia, calcium, and phosphorus imbalance), many children continue to grow poorly. Therefore, three different dialysis modalities, continuous ambulatory peritoneal dialysis (CAPD), cycler/intermittent peritoneal dialysis (CPD), and hemodialysis (HD), were evaluated with regard to their effects on the growth of children initiating dialysis and remaining on that modality for 6-12 months. Growth was best for children undergoing CAPD when compared with the other two modalities with regard to the following growth parameters: incremental height standard deviation score for chronological age [-0.55 +/- 2.06 vs. -1.69 +/- 1.22 for CPD (P < 0.05) and -1.80 +/- 1.13 for HD (P < 0.05)]; incremental height standard deviation score for bone age [-1.68 +/- 1.71 vs. -2.45 +/- 1.43 for CPD (P = NS) and -2.03 +/- 1.28 for HD (P = NS)]; change in height standard deviation score during the dialysis period [0.00 +/- 0.67 vs. -0.15 +/- .29 for CPD (P = NS) and -0.23 +/- .23 for HD (P = NS)]. The reasons why growth appears to be best in children receiving CAPD may be related to its metabolic benefits: lower levels of uremia, as reflected by the blood urea nitrogen [50 +/- 12 vs. 69 +/- 16 mg/dl for CPD (P < 0.5) and 89 +/- 17 for HD (P < 0.05)], improved metabolic acidosis, as indicated by a higher serum bicarbonate concentration [24 +/- 2 mEq/l vs. 22 +/- 2 for CPD (P < 0.05) and 21 +/- 2 for HD (P < 0.05)]. In addition, children undergoing CAPD receive significant supplemental calories from the glucose absorbed during dialysis. CAPD, and possibly, other types of prolonged-dwell daily peritoneal dialysis appear to be most beneficial for growth, which may be of particular importance for the smaller child undergoing dialysis while awaiting transplantation.

Adolescent↗
PubMed1978

Abolition of plasma growth hormone response to stress and of the circadian rhythm in pituitary-adrenal function in female rats with preoptic-anterior hypothalamic lesions.

The chronic effects of lesions placed in the medial or lateral preoptic-anterior hypothalamic area (PO-AHA) on plasma growth hormone (GH) and corticosterone (cpd B) responses to stress, non-stress levels of these hormones and somatic growth were studied in adult female rats. Responses to 3-min immobilization--blood withdrawal stress uere tested at 2 and 11 weeks and to 3-min ether--blood withdrawal at 5 weeks after surgery. Non-stress plasma GH levels were further assessed in serial blood samples collected at 4-h intervals during a 24-h period at 15 weeks, and cpd B levels were studied similarly for a 44-h period at 22 weeks. Rats with lateral PO-AHA lesions showed normal GH suppression and cpd B elevation in response to both stress procedures. In contrast, neither stressor suppressed plasma GH levels in rats with medial PO-AHA lesions, but both produced normal increments in cpd B. The 24-h mean non-stress level of plasma GH in the group with medial lesions was higher and that of the group with lateral lesions was lower than those of controls. Cpd B levels were reduced during the 44-h study, and circadian rhythmicity was abolished in rats with medial lesions. The group with lateral lesions showed normal rhythmicity in plasma cpd B levels, Linear growth was not affected in either lesioned group. The GH findings suggest that the medial but not lateral PO-AHA is essential for stress-induced inhibition of GH secretion and that this region may exert a tonic inhibitory influence on non-stress GH secretion. The data also suggest that the medial PO-AHA is importantly involved in producing the circadian rhythm in pituitary-adrenal function.

Animals↗
PubMed1993

Unique reaction of a barley peroxidase with hydrogen peroxide.

The reaction of barley peroxidase BP 1 with H2O2 is markedly different from that of other peroxidases. Saturation kinetics and a strong pH dependence over the accessible pH range from 3.09 to 5.08 are observed. At pH 3.8, native BP 1 has maxima at 401, 498 and 635 nm, cpd 1 at 403 nm, and cpd II at 407 and 521 nm with a shoulder at 553 nm. Both cpds I and II appear to be incompletely formed. Isosbestic points between native BP 1 and cpd I occur at 365 and 416 nm, while an isosbestic point in the Soret region between cpd I and cpd II has been observed at 410 nm. Between cpd II and a not yet identified intermediate isosbestic points have been observed at 408, 455 and 526 nm.

Hordeum↗
PubMed1996

Fine structure mapping of DNA repair within a 100 kb genomic region in Chinese hamster ovary cells.

We have investigated at a high level of resolution the repair of cyclobutane pyrimidine dimers (CPD) in a large amplified genomic region in Chinese hamster ovary B11 cells. We found strand selective repair in DNA fragments within two active genes, DHFR and an unknown gene adjacent to DHFR. These genes generate divergent transcripts from the same promoter region; their transcribed strands were virtually free of CPD within 24 h after irradiation with 10 j/m2 of ultraviolet light (254nm), while their non-transcribed strands were poorly repaired. We also examined the repair of CPD in three DNA fragments within a 50 kb region downstream of DHFR, in which two origins of replication flanking a matrix attachment site have been characterized from independently derived cell lines with amplified DHFR domains; repair of CPD in this non-transcribed region was similarly poor in both DNA strands. Transcription-coupled repair of CPD in the DHFR gene exhibited the same proficiency throughout the transcription unit: analysis of the efficiency of removal of CPD over time revealed no differences between repair in the 5' and the 3' ends of the DHFR gene. Implications for mechanisms of transcription-coupled repair are discussed.

Animals↗
PubMed1996

Comparison of the rate of excision of major UV photoproducts in the strands of the human HPRT gene of normal and xeroderma pigmentosum variant cells.

Xeroderma pigmentosum (XP) variant patients are genetically predisposed to sunlight-induced skin cancer. Fibroblasts from such patients are extremely sensitive to mutations induced by UV radiation, and the spectrum of mutations induced in their hypoxanthine phosphoribosyltransferase (HPRT) gene differs significantly from that seen in normal cells. To determine if this UV hypermutability reflects abnormally slow excision repair of cyclobutane pyrimidine dimers (CPD) or 6-4 pyrimidine-pyrimidones (6-4s) in that gene, we synchronized XP variant and normal fibroblasts, irradiated them in early G1-phase, 12 or more hours prior to the scheduled onset of S phase, harvested them immediately or after allowing various times for repair, and analyzed the DNA for photoproducts in the HPRT gene, using quantitative Southern blotting. To incise the DNA at CPD, we used T4 endonuclease V; to incise at 6-4s, we first used photolyase and UV365nm to reverse CPD and then UvrABC excinuclease. Excision of CPD was rapid, preferential, and strand-specific, but there was no significant difference in rate between the two kinds of cells. The half life was 4 h in the transcribed strand of the gene and 6.5 h in the nontranscribed strand. For excision of CPD in the genome overall, this value is 12 h. Excision of 6-4s from either strand of the HPRT gene was extremely rapid and preferential in both kinds of cells, with a half life of approximately 30 min. The results indicate that the UV hypermutability of the XP variant cells cannot be caused by slower rates of repair of CPD and/or 6-4s in the target gene for mutagenesis.

Blotting, Southern↗
PubMed2005

DNA polymerases eta and kappa are responsible for error-free translesion DNA synthesis activity over a cis-syn thymine dimer in Xenopus laevis oocyte extracts.

In translesion synthesis (TLS), specialized DNA polymerases (pols) facilitate progression of replication forks stalled by DNA damage. Although multiple TLS pols have been identified in eukaryotes, little is known about endogenous TLS pols and their relative contributions to TLS in vivo because of their low cellular abundance. Taking advantage of Xenopus laevis oocyte cells, with their extraordinary size and abundant enzymes involved in DNA metabolism, we have identified and characterized endogenous TLS pols for DNA damage induced by ultraviolet (UV) irradiation. We designed a TLS assay which monitors primer elongation on a synthetic oligomer template over a single UV-induced lesion, either a cys-syn cyclobutane pyrimidine dimer (CPD) or a pyrimidine (6-4) pyrimidone photoproduct. Four distinct TLS activities (TLS1-TLS4) were identified in X. laevis oocyte extracts, using three template/primer (T/P) DNA substrates having various sites at which primer extension is initiated relative to the lesion. TLS1 and TLS2 activities appear to be sequence-dependent. TLS3 and TLS4 extended the primers over the CPD in an error-free manner irrespective of sequence context. Base insertion opposite the CPD of the T/P substrate in which the 3'-end of the primer is placed one base upstream of the lesion was observed only with TLS3. TLS3 and TLS4 showed primer extension with similar efficiencies on the T/P substrate whose 3'-primer terminal dinucleotide (AA) was complementary to the CPD lesion. Investigations with antibodies and recombinant pols revealed that TLS3 and TLS4 were most likely attributable to pol eta and pol kappa, respectively. These results indicate that error-free insertion in CPD bypass is due mainly to pol eta (TLS3) in the extracts, and suggest that pol kappa (TLS4) may assist pol eta (TLS3) in error-free extension during CPD bypass.

Animals↗