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Biomedical subjects

S Linn

Publications and source records attributed to S Linn.

At least 55 records · Page 3Linked to original sources

The sky is a limit: errors in prehospital diagnosis by flight physicians.

The medical records and air evacuation reports of 186 trauma patients were examined to determine the type and characteristics of missed diagnoses. More than 35% of all cases of hypovolemic shock were not identified, nor were two cases of respiratory distress. Although unconsciousness was always identified correctly, almost 7% of all cases with partial unconsciousness were not recorded. Of 443 diagnoses, 337 were correctly recorded by the flight physician, slightly more than 76%. The flight physicians missed 10 critical diagnoses, all of which were feasible, 56 important diagnoses, 42 of which were feasible, and 40 relatively marginal diagnoses, 27 of which were feasible. Injuries to the head, face, and limbs were usually diagnosed correctly, and were missed only in a few cases. Of considerable clinical relevance was the observation that flight physicians missed a significant number of critical and important feasible diagnoses of five types: (1) more than half of all feasible diagnoses in the eyes; (2) a third of feasible diagnoses of cervical spine injuries; and a significant percentage of injuries to the (3) abdomen, (4) chest, and (5) pelvis. Blunt diagnoses were missed more often than penetrating injuries. Feasible diagnoses were missed in two of the four cases of paralysis, approximately one third of all crush injuries, and one quarter of all fractures. This study illuminates preventable errors of physicians during air evacuation and indicates particular types of serious, feasible diagnoses that flight physicians are prone to miss. Medicine in the sky may pose limits to our diagnostic abilities but the limits could be pushed further.

Adolescent↗

The application of the macular photocoagulation study eligibility criteria for laser treatment in age-related macular degeneration.

BACKGROUND AND OBJECTIVE: To analyze the application of the Macular Photocoagulation Study eligibility criteria for laser photocoagulation of choroidal neovascularization, in view of the expansion of these criteria in recent years. PATIENTS AND METHODS: The authors prospectively analyzed 50 eyes of 47 consecutive patients with exudative age-related macular degeneration (AMD) to determine their suitability for treatment. RESULTS: Fifteen eyes (30%) were found to be suitable for laser photocoagulation. Patients eligible for treatment were more likely to have experienced visual symptoms for a months or less (P = .006), to have a visual acuity of 20/200 or better (P = .009), and to be younger in age (P = .02). Visual symptoms experienced for a month or less were more prevalent in extrafoveal exudative lesions compared with the subfoveal type (P = .01) CONCLUSIONS: Despite recent advances, laser photocoagulation still can be applied only to a minority of the patients with neovascular AMD. Prompt ocular examination following the onset of visual symptoms is essential.

Aged↗

Mutations specific to the xeroderma pigmentosum group E Ddb- phenotype.

The activity of a damage-specific DNA-binding protein (DDB) is absent from a subset, Ddb-, of cell strains from patients with xeroderma pigmentosum group E (XP-E). DDB is a heterodimer of 127-kDa and 48-kDa subunits. We have now identified single-base mutations in the gene of the 48-kDa subunit in cells from the three known Ddb- individuals, but not in XP-E strains that have the activity. An A --> G transition causes a K244E change in XP82TO and a G --> A transition causes an R273H change in XP2RO and XP3RO. No mutations were found in the cDNA of the 127-kDa subunit. Overexpression of p48 in insect cells greatly increases DDB activity in the cells, especially if p127 is jointly overexpressed. These results demonstrate that p48 is required for DNA binding activity, but at the same time necessitate further definition of the genetic basis of XP group E.

Animals↗

Fe2+, Fe3+, and oxygen react with DNA-derived radicals formed during iron-mediated Fenton reactions.

Oxidative DNA damage is decreased by the presence of O2 during Fe(2+)-mediated Fenton reactions when H2O2 is in excess. During these reactions, the presence of DNA increases H2O2 consumption relative to Fe2+ consumption under anaerobic conditions, but decreases H2O2 consumption relative to Fe2+ consumption under aerobic conditions. The pseudobimolecular rate constant of H2O2 consumption is the same under both conditions, however, indicating that the presence of DNA affects the oxidation and/or reduction of the iron pool. To understand the basis of these effects, DNA was replaced with ethanol as a model compound. Computer simulations of Fe2+ and H2O2 consumption were experimentally verified and allowed identification of the predominant reactions leading to the changes in stoichiometry. Based upon these results and upon qualitative and quantitative differences in DNA damages between aerobic and anaerobic conditions, it was concluded that, in the presence of DNA, Fe3+ is reduced by some DNA radicals. However, if O2 is present, these radicals react instead with O2 and the product of these reactions can then oxidize Fe2+. Mechanisms proposed for the alteration by O2 of products from dC- and dG-containing substrates after exposure to Fe and H2O2 fit these general schemes. These results provide another distinction between DNA damage caused by ionizing radiation and that caused by Fenton reactions.

Acetaldehyde↗

Oxidative damage to DNA constituents by iron-mediated fenton reactions. The deoxycytidine family.

Damage by iron-mediated Fenton reactions under aerobic or anaerobic conditions to deoxycytidine, deoxycytidine-5'-monophosphate, d-CpC, d-CpCpC, and dCMP residues in DNA resulted in at least 26 distinguishable products. Of these, 24 were identified by high performance liquid chromatography retention times, radiolabeling, UV absorption spectra, chemical synthesis, fast atom bombardment mass spectrometry, high resolution fast atom bombardment mass spectrometry, and/or NMR. The nature of the products was qualitatively similar for each substrate except for d-CpC (and possibly d-CpCpC) under anaerobic conditions for which 5-hydroxy-deoxycytidine was uniquely present and 1-carbamoyl-1-carboxy-4-(2-deoxy-beta-D-erythropentofuranosyl) glycinamide was uniquely absent. Damage to dC, d-CpC, and d-CpCpC but not to dCMP or DNA was largely quenched by ethanol, indicating that iron is strongly associated only with dCMP and DNA. The presence of oxygen had little effect with dC or dCMP but had quantitative and qualitative effects with d-CpC and a significantly quantitative but not a qualitative effect with DNA. NADH could drive the Fenton reaction to cause damage to the dC family in vitro, consistent with a previous proposal that NADH was the reducing agent for the Fenton reaction in vivo (Imlay, J.A., and Linn, S. (1988) Science 240, 1302-1309). Finally, the damage spectrum of the dC family by the Fenton reaction is compared with that by ionizing radiation and chemical mechanisms leading to the formation of the 24 identified products are proposed.

Chromatography, High Pressure Liquid↗

Oxidative damage to DNA constituents by iron-mediated fenton reactions. The deoxyguanosine family.

2'-Deoxyguanosine, 3'-dGMP, 5'-dGMP, d-GpG, or double-stranded DNA were exposed to H2O2 in the presence of Fe2+ under anaerobic conditions or under aerobic conditions in the presence of Fe3+, Fe2+, Fe2+/NADH, or Fe3+/NADH with and without ethanol. The products were enzymatically digested to nucleosides, separated by high performance liquid chromatography (HPLC), quantified, and characterized by HPLC retention time, radiolabeling, UV absorbance spectrometry, NMR, and mass spectrometry. 20 products, constituting 78-81% of the damage, were distinguished from aerobic reactions of Fe2+/H2O2 with dG and dGMP, 16 of which were identified. The product spectra from dG, 3'-dGMP, and 5'-dGMP differ from one another, and the spectrum of the 5' nucleoside of d-GpG differs from that of the 3' nucleoside. 7, 8-Dihydro-8-oxo-2'-deoxyguanosine is the most abundant DNA-bound product aside from abasic sites, and its formation was more closely analyzed. Both NADH, which can reduce Fe3+, and ethanol, which can scavenge some free radicals, change the product profiles. Damage enhancement by NADH follows the sequence dG < d-GpG < 3'-dGMP < 5'-dGMP < DNA; the reverse sequence is observed for ethanol quenching. This sequence of susceptibility and the product differences seen for the 3' and 5' phosphate may reflect localization of iron and the damaging radicals upon the substrate.

Chromatography, High Pressure Liquid↗

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↗

A human RNA polymerase II complex associated with SRB and DNA-repair proteins.

We report here the isolation of a human RNA polymerase II complex containing a subset of the basal transcription factors and the human homologues of the yeast SRB (for suppressors of RNA polymerase B) proteins. The complex contains transcriptional coactivators and increases the activation of transcription. In addition, some components of the RNA polymerase II complex participate in DNA repair.

Chromatography, Affinity↗

Analysis of the mammalian recombination protein complex RC-1.

Based on a novel cell-free assay for DNA recombination, we previously reported the purification and initial characterization of RC-1, a protein complex catalyzing the recombinational repair of deletions and gaps. RC-1 was isolated from calf thymus nuclear extracts and shown to copurify with several enzymatic activities, among them a DNA polymerase. Here, additional evidence is reported identifying the polymerase as DNA polymerase epsilon. Furthermore, a novel DNA structure-dependent endonuclease associated with RC-1 was observed, which recognizes and cleaves branched DNA substrates at specific sites. Implications of this endonuclease activity for the recombination reaction are discussed.

Base Sequence↗

Overdiagnosis of 21-hydroxylase late onset congenital adrenal hyperplasia: correlation of corticotropin test and human leukocyte antigen typing.

OBJECTIVE: To evaluate the reliability of the ACTH test as a means for detection of late onset congenital adrenal hyperplasia (CAH) and discriminating it from polycystic ovary syndrome (PCOS), by repeating the test after 6 months of cyproterone acetate and ethinyl E2 treatment. DESIGN: Follow-up comparison study. SETTING: Reproductive Endocrinology in an university tertiary center. PATIENTS: Thirty-one young women with hirsutism, oligoamenorrhea, and acne, 21 of them detected as late onset CAH, and 10 as non-late onset CAH (PCOS). INTERVENTION: Cyproterone acetate and ethinyl E2 treatment for > or = 6 months. The ACTH test, before and after 6 months of cyproterone acetate + ethinyl E2 treatment, and human leukocyte antigen (HLA) typing. MAIN OUTCOME MEASURE: The ACTH test interpretation correlated to HLA typing. RESULTS: By repeating the ACTH stimulation test in the 31 women (after cyproterone acetate + ethinyl E2 administration), we found a diminution in the rate of accumulation of 17 alpha-hydroxyprogesterone (delta 17-OHP) + P, in all 21-hydroxylase late onset CAH cases. As a result of treatment with cyproterone acetate + ethinyl E2, a decrease in the accumulation rate of 17-OHP + P, below the discriminative value for late onset CAH (6.5 ng/dL per minute), was noted among 12 of 21 women defined primarily as late onset CAH. Among the nine other women, a decrease in the accumulation rate of 17-OHP + P was noted, however not < 6.5 ng/dL per minute. CONCLUSIONS: The interpretation of delta 17-OHP + P for the diagnosis of late onset CAH may be too sensitive as to the correct clinical diagnosis of late onset CAH. By repeating the ACTH test after 6 months of treatment with cyproterone acetate-ethinyl E2, specificity and accuracy may be improved.

17-alpha-Hydroxyprogesterone↗

Prevention of irreversible chemotherapy-induced ovarian damage in young women with lymphoma by a gonadotrophin-releasing hormone agonist in parallel to chemotherapy.

To examine whether the concomitant administration of a gonadotrophin-releasing hormone agonist (GnRHa) during combination chemotherapy to young women with lymphoma may facilitate preservation of gonadal function, a prospective clinical protocol was undertaken in 18 cycling women with lymphoma, aged 15-40 years. Thirteen patients suffered from Hodgkin disease (HD) and 5 from non-Hodgkin lymphoma. After informed consent a monthly injection of depot D-TRP6-GnRHa was administered for a maximum of 6 months starting prior to chemotherapy. Most of these patients (15/18) were treated with the MOPP/ABV(D) combination chemotherapy followed by mantle field irradiation in 10 patients. Hormonal profile [luteinizing hormone (LH), follicle stimulating hormone (FSH), oestradiol, testosterone, progesterone, insulin-like growth factor (IGF)-1, prolactin] was taken before the GnRHa/chemotherapy co-treatment, and monthly thereafter until resuming spontaneous ovulation and menses. This group of prospectively treated lymphoma patients was compared to a matched control group of 18 women (aged 17-40 years) who have been treated with chemotherapy, mostly MOPP/ABV (14/18), with (11) or without (7) mantle field radiotherapy. Fourteen had Hodgkin's and four non-Hodgkin's lymphoma. Gonadal function was determined clinically, hormonally (LH, FSH, oestradiol, progesterone), and sonographically. Two of the patients in each group died from refractory disease. Of the remaining 16 patients, 15 (93.7%) resumed spontaneous ovulation and menses within 3-8 months of termination of the combined chemotherapy/GnRHa co-treatment. In contrast, only seven (39%) of the 18 similarly treated patients in the control group (chemotherapy without GnRHa) resumed ovarian cyclic activity (regular menses). The other 11 experienced premature ovarian failure (POF) (61%). Out preliminary data suggest a possible significant protective effect of GnRHa co-treatment with chemotherapy from irreversible ovarian damage (POF).

Adolescent↗

Cisplatin-induced alterations in the expression of the mRNAs for UV-damage recognition protein.

Enhanced DNA repair is believed to be an important mechanism of the cisplatin-resistant phenotype. UV-damage recognition protein (UV-DRP) recognizes and binds to DNA lesions and may play a role in DNA nucleotide excision repair and/or replicative bypass (which is associated with post-replication repair). Potential alternations in the expression of mRNAs for UV-DRP were analyzed in this study. Two pairs of parental and cisplatin-resistant human ovarian carcinoma cell lines were utilized. Gene expression level was assessed by northern blot hybridization. No alterations in mRNA levels for the large subunit of UV-DRP were found following cisplatin treatment, whereas mRNA levels for the small subunit of UV-DRP were induced up to 4.5-fold. The time-course and concentration-response of this induction corresponded to the previously reported increase in the UV-DRP binding activity, as measured by gel shift assay. UV-DRP binding activity in cell extracts corresponds to expression of small subunit mRNA but not to expression of large subunit mRNA. These data suggest that the small subunit may be limiting for UV-DRP activity.

Antineoplastic Agents↗

Invasion of selectively permeable sea urchin embryo basement membranes by metastatic tumor cells, but not by their normal counterparts.

The selectively permeable basement membranes and the associated extracellular matrix of sea urchin embryos can be obtained intact. Their exterior surfaces have been used as invasion substrates for metastatic melanoma, squamous cell carcinoma, and fibrosarcoma cells, for primary squamous cell carcinoma cells, and for neonatal melanocytes, fibroblasts, and keratinocytes. About 18% of all metastatic tumor cells placed in contact with sea urchin embryo basement membranes and their associated extracellular matrix invaded them. About 4% of the cells of a primary squamous cell carcinoma, which later metastasized, invaded these substrates. As expected, neonatal melanocytes, keratinocytes, and fibroblasts failed to invade; however, melanocytes treated with scatter factor (hepatocyte growth factor) invaded as efficiently as metastatic tumor cells. This suggests that the lack of invasion by epidermal melanocytes is not due to irreversible differentiation to a noninvasive phenotype. Invasion time courses showed that the metastatic cells tested reached their maximal invasion frequencies in 4 h; thus, invasion of these substrates is rapid and efficient. This suggests that molecules participating in basement membrane recognition and invasion have been functionally conserved during the time separating vertebrates from invertebrates and that their constitutive activity may allow metastatic cells to escape their tissues of origin.

Animals↗

Chromosomal localization and cDNA cloning of the genes (DDB1 and DDB2) for the p127 and p48 subunits of a human damage-specific DNA binding protein.

DDB is a damage-specific DNA binding protein whose binding activity is absent from a minority of cell strains from individuals with xeroderma pigmentosum Group E, a human hereditary disease characterized by defective nucleotide excision DNA repair and an increased incidence of skin cancer. The binding activity from HeLa cells is associated with polypeptides of M(r) 124,000 and 41,000 as determined by SDS-polyacrylamide gels. This report describes the isolation of full-length human cDNAs encoding each polypeptide of DDB. The predicted peptide molecular masses based on open reading frames are 127,000 and 48,000. When expressed in an in vitro rabbit reticulocyte system, the p48 subunit migrates with an M(r) of 41 kDa on SDS-polyacrylamide gels, similarly to the peptide purified from HeLa cells. There is no significant homology between the derived p48 peptide sequence and any proteins in current databases, and the derived peptide sequence of p127 has homology only with the monkey DDB p127 (98% nucleotide identity and only one conserved amino acid substitution). Using a fluorescence in situ hybridization technique, the DDB p127 locus (DDB1) was assigned to the chromosomal location 11q12-q13, and the DDB p48 locus (DDB2) to 11p11-p12.

Amino Acid Sequence↗

Implication of mammalian ribosomal protein S3 in the processing of DNA damage.

A human apurinic/apyrimidinic endonuclease activity, called AP endonuclease I, is missing from or altered specifically in cells cultured from Xeroderma pigmentosum group-D individuals (XP-D cells) (Kuhnlein, U., Lee, B., Penhoet, E. E., and Linn, S. (1978) Nucleic Acids Res. 5,951-960). We have now observed that another nuclease activity, UV endonuclease III, is similarly not detected in XP-D cells and is inseparable from the AP endonuclease I activity. This activity preferentially cleaves the phosphodiester backbone of heavily ultraviolet-irradiated DNA at unknown lesions as well as at one of the phosphodiester bonds within a cyclobutane pyrimidine dimer. The nuclease activities have been purified from mouse cells to yield a peptide of M(r) = 32,000, whose sequence indicates identity with ribosomal protein S3. The nuclease activities all cross-react with immunopurified antibody directed against authentic rat ribosomal protein S3, and, upon expression in Escherichia coli of a cloned rat cDNA for ribosomal protein S3, each of the activities was recovered and was indistinguishable from those of the mammalian UV endonuclease III. Moreover, the protein expressed in E. coli and its activities cross-react with the rat protein antibody. Ribosomal protein S3 contains a potential nuclear localization signal, and the protein isolated as a nuclease also has a glycosylation pattern consistent with a nuclear localization as determined by lectin binding. The unexpected role of a ribosomal protein in DNA damage processing and the unexplained inability to detect the nuclease activities in extracts from XP-D cells are discussed.

Amino Acid Sequence↗

Further characterization of HeLa DNA polymerase epsilon.

DNA polymerase epsilon (pol epsilon) from HeLa cells was purified to near homogeneity, utilizing Mono S fast protein liquid chromatography for complete separation from pol alpha. The purified pol epsilon preparation showed two polypeptides of > 200 and 55 kDa and a small amount of active 122-kDa proteolysis product on denaturing polyacrylamide gels. Pol epsilon (as well as pols alpha and delta) is optimally active in 100-150 mM potassium glutamate and 15 mM MgCl2. Replication factors RF-A and RF-C, proliferating cell nuclear antigen, and Escherichia coli single-stranded DNA binding protein showed no significant effect on this preparation's pol epsilon activity, processivity, or substrate specificity. The size of the pol epsilon transcript for the catalytic subunit (> 200 kDa) was investigated in both normal human fibroblasts and HeLa cells. A 7.7-kilobase transcript was detected which was 5-16-fold more prevalent in proliferating than in quiescent HeLa cells. No significant difference in the level of pol epsilon transcript in HeLa cells or fibroblasts was seen after ultraviolet irradiation. Mouse polyclonal antiserum was produced to a 144-amino acid fragment of pol epsilon fused to staphylococcal protein A. This non-neutralizing polyclonal antiserum specifically recognized the catalytic subunit of pol epsilon by immunoblotting, but not that of pol alpha, beta, or delta. In addition, mouse polyclonal antiserum raised against column-purified pol epsilon was able to recognize and to neutralize pol epsilon, and a mouse monoclonal antibody was raised which was able to recognize specifically the catalytic subunit of pol epsilon.

Animals↗

Purification of all forms of HeLa cell mitochondrial DNA and assessment of damage to it caused by hydrogen peroxide treatment of mitochondria or cells.

A purification scheme for mitochondrial DNA (mtDNA) was designed which maximized the yield of all forms of the DNA while minimizing damage to the DNA during its isolation. Treatment of intact mitochondria with DNase I removed nuclear DNA and the avoidance of phenol and the isolation by CsCl density gradients in the absence of ethidium bromide and subsequent detection by Southern Hydridization dot-blots minimized DNA damage. Four different mtDNA forms free of apparent nuclear DNA were obtained: closed circular (I), open circular (II), linear (III), and a large multimer complex (C) which were characterized by agarose gel electrophoresis and electron microscopy. Using this procedure, mtDNA was obtained from both whole cells or intact mitochondria treated with H2O2. Significant fragmentation was observed after treatment at 37 degrees C, but not at 0 degrees C, and more damage was observed when treating whole cells than isolated mitochondria. Very low levels of 8-hydroxydeoxyguanosine were observed in all cases. However, at doses of H2O2 which were just lethal, neither increased DNA damage nor inactivation of cytochrome c oxidase was observed.

Base Sequence↗