Biomedical subjects

G Siegal

Publications and source records attributed to G Siegal.

25 records · Page 2Linked to original sources

PubMed1992

DNA helicase E and DNA polymerase epsilon functionally interact for displacement synthesis.

A functional interaction between DNA helicase E and DNA polymerase epsilon from calf thymus has been detected which results in the extension of an upstream 3' OH through a downstream primer to the end of a synthetic template. DNA synthesis resulting in full-length extension products was dependent on the addition of DNA helicase E and hydrolysis of ATP, suggesting that displacement of the downstream primer was required. Identical reactions using DNA polymerases alpha and delta in place of DNA polymerase epsilon showed no full-length products dependent on helicase E, indicating that polymerases alpha and delta were incapable of functionally interacting with the helicase. The reaction leading to full-length extension products was time dependent and dependent on the concentration of added polymerase epsilon and helicase E. Exonucleolytic degradation of the downstream primer, or ligation of the downstream primer to the upstream 3' OH, were not responsible for the full-length products observed. Displacement of the downstream primer by DNA helicase E was not affected by the addition of polymerase epsilon to the reactions. Template dilution experiments demonstrated that DNA polymerase epsilon and helicase E were acting in concert to perform displacement synthesis. Additional evidence for functional coordination was obtained by demonstration that DNA helicase E stimulated DNA polymerase epsilon in a standard DNA synthetic assay using dA3000.dT16 as the template-primer. The results presented are consistent with the hypothesis that DNA helicase E and DNA polymerase epsilon are capable of coordinated activities that result in displacement synthesis. A functional interaction of this sort may be involved at the eukaryotic replication fork or in DNA repair.

Adenosine Triphosphate↗

PubMed1992

A novel DNA helicase from calf thymus.

We report the purification and characterization of a novel DNA helicase from calf thymus tissue. This enzyme partially copurifies with DNA polymerase epsilon* through many of the chromatographic procedures used to isolate it. The enzyme contains an intrinsic DNA-dependent ATPase activity. It can displace short oligonucleotides annealed to long single stranded substrates, in an ATP-dependent reaction. Use of this assay indicates that the DNA helicase translocates in a 3' to 5' direction with respect to the substrate strand to which it is bound. Maximal efficiency of displacement is accomplished by hydrolysis of (d)ATP as cofactor, however, (d)CTP can also be utilized resulting in a 5-fold decrease in the level of displacement. Displacement activity is enhanced by the presence of saturating amounts of Escherichia coli single stranded DNA-binding protein, not affected by the presence of phage T4 gene 32 protein, and inhibited by human replication factor A. The DNA helicase has a molecular mass of approximately 104 kDa as measured by denaturing gel electrophoresis, and an S value of 5.4 obtained from glycerol gradient sedimentation. Direct [alpha-32P]ATP cross-linking labels a protein of molecular mass approximately 105 kDa, providing further evidence that this polypeptide contains the helicase active site. In view of the differences in the properties of this helicase from four others recently identified in calf and designated A through D, we propose the name helicase E.

Adenosine Triphosphate↗

PubMed1992

Structural relationships between two forms of DNA polymerase epsilon from calf thymus.

We previously reported purification of two forms of DNA polymerase epsilon from calf thymus (Crute, J. J., Wahl, A. F., and Bambara, R. A. (1986) Biochemistry 25, 26-36). We have now used the "polymerase trap" photolabeling method to identify the polypeptides containing the polymerase active site in each enzyme preparation. The molecular mass of these polypeptides are 210 and 145 kDa for the polymerases now designated epsilon and epsilon*, respectively. Renaturation of polymerase activity from denaturing gel electrophoresis corroborates the polymerase trap results. Photolabeling of polymerase fractions suggests that the smaller subunit is derived by proteolysis of the larger subunit during purification. Native sedimentation coefficient measurements of polymerase-containing column fractions further suggest a precursor/product relationship between the two polymerases. Response of polymerization activity to a battery of inhibitors normally used to distinguish mammalian nuclear DNA polymerases was found to be essentially identical for polymerases epsilon, epsilon*, and the epsilon* generated in fractions initially containing epsilon. These latter results demonstrate that the loss of the protease-sensitive domain of the active site subunit does not affect catalytic function as measured in a standard DNA polymerase assay. The sole apparent functional difference observed here between the epsilon and epsilon* forms is evidence that only the full-length epsilon form can be directly photocrosslinked to dATP, independent of DNA synthesis. Photolabeling of the post-microsomal supernatant fraction from thymus glands obtained from fetal calves reveals the presence of both the epsilon and epsilon* polypeptide.

Animals↗

PubMed1992

Breast masses in adolescent females.

In a retrospective study of 38 adolescent females operated on for mammary masses, 42 fibroadenomata (FA), 2 fibrous scars around abscesses, and one papilloma were found. All were postmenarchal. Clinical and pathological features were reviewed and the patients reexamined or interviewed. FA were mostly asymptomatic and removed a few weeks after incidental discovery. They were often seen in Jewish girls of parents born in North Africa and the Middle East, and were more frequently located in the outer superior quadrant of the left breast. New FA appeared in almost half of the cases later on in the same breast, as well as in the other breast, or bilaterally. Necrosis occurred in three FA and was unrelated to the size of the tumor. There was no correlation between the size of FA, multiplicity, or necrosis, nor between increased stromal or epithelial atypia, cellularity, or mitotic activity. One explanation for the high rate of multiplicity might be related to this neoplasm occurring in an actively developing organ under hormonal control. There was no increased breast carcinoma apparent in the family history. Therefore, except in cases of fast-growing tumors, a conservative approach is recommended because of the frequency of multiple FA as well as aesthetical postoperative complications.

Adenofibroma↗

PubMed1992

Fibronectin as a component of pseudophakic acellular membranes.

Avidin-biotin immunohistochemical staining for fibronectin was used to study 17 aphakic intraocular lens implanted globes and 12 explanted intraocular lenses. Fibronectin was identified as a component of acellular pseudophakic membranes and was also observed within lens epithelial cells and macrophages.

Cataract Extraction↗

PubMed1989

Tumor necrosis factor-mediated cytotoxicity by tumor-associated macrophages.

We have directly demonstrated that macrophages present within solid EMT6 mammary tumors (of BALB/c origin) produce TNF-alpha (TNF). These tumor-associated macrophages lysed WEHI-164, a TNF-sensitive cell line, very efficiently. This cytotoxicity was abrogated in the presence of anti-TNF antisera. In contrast, EMT6 cells, the tumor from which the macrophages were obtained, were not effectively lysed by the macrophages and were 100-fold less sensitive to lysis by recombinant mouse TNF. Thus, marked heterogeneity exists among tumors regarding sensitivity to TNF-mediated cytotoxicity. Similarly, macrophages which infiltrate into EMT6 multicellular spheroids implanted into the peritoneal cavity as well as free cells within the cavity exhibited TNF-mediated cytotoxicity of WEHI-164 cells, but failed to lyse EMT6 cells. The kinetics of lysis by these cells was similar to that of recombinant mouse TNF.

Animals↗

PubMed1986

Endometrial stromal cells in culture: an attempt to understand the genesis and biologic activity of uterine sarcomas.

The genesis of uterine sarcomas in vivo has been difficult to elucidate. The reasons are multifactorial and are compounded by the rarity of these tumors. In an effort to understand the biologic activity of this disease, normal endometrial tissue was subjected to tissue culture, histochemical study, and hormonal manipulation. Protein markers were used to differentiate endometrial epithelium from endometrial stromal cells in culture. The endometrial stromal cells grew rapidly following subculturing and were responsive to hormonal manipulation. When the stromal cells in culture were treated with the carcinogen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), the resultant morphologic changes mimicked uterine sarcomas grown in culture. These changes appeared to occur in a sequential manner, somewhat analogous to changes occurring in the endometrial epithelium as it undergoes transformation to endometrial adenocarcinoma. These studies, therefore, may serve as a model system in understanding the genesis and biologic activity of uterine sarcomas.

Alkaline Phosphatase↗