BackgroundIn humans, serotonin has typically been investigated as a neurotransmitter. However, serotonin also functions as a hormone across animal phyla, including those lacking an organized central nervous system. This hormonal action allows serotonin to have physiological consequences in systems outside the central nervous system. Fluctuations in estrogen levels over the lifespan and during ovarian cycles cause predictable changes in serotonin systems in female mammals.DiscussionWe hypothesize that some of the physiological effects attributed to estrogen may be a consequence of estrogen-related changes in serotonin efficacy and receptor distribution. Here, we integrate data from endocrinology, molecular biology, neuroscience, and epidemiology to propose that serotonin may mediate the effects of estrogen. In the central nervous system, estrogen influences pain transmission, headache, dizziness, nausea, and depression, all of which are known to be a consequence of serotonergic signaling. Outside of the central nervous system, estrogen produces changes in bone density, vascular function, and immune cell self-recognition and activation that are consistent with serotonin's effects. For breast cancer risk, our hypothesis predicts heretofore unexplained observations of the opposing effects of obesity pre- and post-menopause and the increase following treatment with hormone replacement therapy using medroxyprogesterone.SummarySerotonergic mediation of estrogen has important clinical implications and warrants further evaluation.

Site-directed mutagenesis was employed to examine the role played by specific surface residues in the activity of cytochrome c peroxidase. The double charge, aspartic acid to lysine, point mutations were constructed at positions 37, 79, and 217 on the surface of cytochrome c peroxidase, sites purported to be within or proximal to the recognition site for cytochrome c in an electron-transfer productive complex formed by the two proteins. The resulting mutant peroxidases were examined for catalytic activity by steady-state measurements and binding affinity by two methods, fluorescence binding titration and cytochrome c affinity chromatography. The cloned peroxidases exhibit similar UV-visible spectra to the wild-type yeast protein, indicating that there are no major structural differences between the cloned peroxidases and the wild-type enzyme. The aspartic acid to lysine mutations at positions 79 and 217 exhibited similar turnover numbers and binding affinities to that seen for the "wild type-like" cloned peroxidase. The same change at position 37 caused more than a 10-fold decrease in both turnover of and binding affinity for cytochrome c. This empirical finding localizes a primary recognition region critical to the dynamic complex. Models from the literature proposing structures for the complex between peroxidase and cytochrome c are discussed in light of these findings.

Walleye dermal sarcoma virus (WDSV) is a complex retrovirus associated with dermal sarcomas of walleye that develop and regress on a seasonal basis. WDSV contains, in addition to gag, pol, and env, three open reading frames (ORFs) designated ORF A, ORF B, and ORF C. The polymerase chain reaction technique was used to amplify and clone cDNAs representing subgenomic viral mRNAs isolated from developing (fall) and regressing (spring) tumors. Nine different singly or multiply spliced viral transcripts were identified and all were found to utilize a common 5' leader sequence. This leader sequence is spliced to the pol/env junction or downstream of env to generate singly spliced transcripts. Multiply spliced transcripts contain the 5' leader, the pol/env junction, and sequences derived from the 3' end of the genome. One multiply spliced transcript was isolated with the potential to encode the full-length ORF A protein. In addition, WDSV produced mRNAs that utilize alternative splice acceptor sites which would allow synthesis of five variant forms of the ORF A protein. In contrast, the ORF B protein is postulated to arise from a singly spliced transcript with the potential to encode the entire open reading frame. Spliced subgenomic transcripts representing ORF C mRNAs were not identified, suggesting that ORF C may be encoded from the full-length viral genomic transcript. We estimate that at least a 100-fold lower amount of the accessory/regulatory subgenomic transcripts exists in developing vs regressing tumors. These results demonstrate that WDSV undergoes an elaborate pattern of mRNA splicing similar to that of other complex retroviruses.

Walleye dermal sarcoma virus (WDSV) is a fish retrovirus associated with the development of tumors in walleyes. We have determined the complete nucleotide sequence of a DNA clone of WDSV, the N-terminal amino acid sequences of the major proteins, and the start site for transcription. The long terminal repeat is 590 bp in length, with the U3 region containing consensus sequences likely to be involved in viral gene expression. A predicted histidyl-tRNA binding site is located 3 nucleotides distal to the 3 end of the long terminal repeat. Virus particles purified by isopycnic sedimentation followed by rate zonal sedimentation showed major polypeptides with molecular sizes of 90, 25, 20, 14, and 10 kDa. N-terminal sequencing of these allowed unambiguous assignment of the small polypeptides as products of the gag gene, including CA and NC, and the large polypeptide as the TM product of env. The 582-amino-acid (aa) Gag protein precursor is predicted to be myristylated as is found for most retroviruses. NC contains a single Cys-His motif like those found in all retroviruses except spumaviruses. The WDSV pro and pol genes are in the same translational reading frame as gag and thus apparently are translated after termination suppression. The env gene encodes a surface (SU) protein of 469 aa predicted to be highly glycosylated and a large transmembrane (TM) protein of 754 aa. The sequence of TM is unusual in that it ends in a very hydrophobic segment of 65 residues containing a single charged residue . Following the env gene are two nonoverlapping long open reading frames of 290 aa (orf-A) and 306 aa (orf-B), neither of which shows significant sequence similarity with known genes. A third open reading frame of 119 aa (orf-C) is located in the leader region preceding gag. The predicted amino acid sequence of reverse transcriptase would place WDSV phylogenetically closest to the murine leukemia virus-related genus of retroviruses. However, other members of this genus do not have accessory genes, suggesting that WDSV acquired orf-A, orf-B, and perhaps orf-C late in its evolution. We hypothesize by analogy with other complex retroviruses that the accessory genes of WDSV function in the regulation of transcription and in RNA processing and also in the induction of walleye dermal sarcoma.

Proliferating cell nuclear antigen (PCNA) is required for DNA homologous recombination (HR), but its exact role is unclear. Here, we investigated the loading of PCNA onto a synthetic D-loop (DL) intermediate of HR and the functional interactions of PCNA with Rad51 recombinase and DNA polymerase (Pol) δ, Pol η, and Pol ζ. PCNA was loaded onto the synthetic DL as efficiently as it was loaded onto a primed DNA substrate. Efficient PCNA loading requires Replication Protein A, which is associated with the displaced ssDNA loop and provides a binding site for the clamp-loader Replication Factor C. Loaded PCNA greatly stimulates DNA synthesis by Pol δ within the DL but does not affect primer recognition by Pol δ. This suggests that the essential role of PCNA in HR is not recruitment of Pol δ to the DL but stimulation of Pol δ to displace a DNA strand during DL extension. Both Pol η and Pol ζ extended the DL more efficiently than Pol δ in the absence of PCNA, but little or no stimulation was observed in the presence of PCNA. Finally, Rad51 inhibited both the loading of PCNA onto the DL and the extension of the DL by Pol δ and Pol η. However, preloaded PCNA on the DL counteracts the Rad51-mediated inhibition of the DL extension. This suggests that the inhibition of postinvasion DNA synthesis by Rad51 occurs mostly at the step of PCNA loading.DNA repair | translesion polymerase | sliding clamp D NA double-strand breaks (DSBs) are introduced into the genome by several factors, including ionizing radiation, mutagenic chemicals, reactive oxygen species, and stalled DNA replication (1). Without appropriate repair, DSBs may lead to cell lethality or cancer (2-4). Homologous recombination (HR) is a widely conserved essential mechanism for high-fidelity repair of DSBs (5-8). HR is a highly coordinated multistep biochemical process, which is most elegantly demonstrated in the yeast Saccharomyces cerevisiae. Briefly, DSB ends are first processed by specialized exonucleases to generate 3′ overhangs (9-11), which are then coated with the ssDNA binding protein Replication Protein A (RPA). The Rad52 recombination mediator interacts with RPA and recruits Rad51 recombinase onto the ssDNA to form a helical nucleoprotein filament (12-15). This Rad51-ssDNA filament mediates strand invasion into a homologous dsDNA (16) to produce a DNA structure that is referred to as the D-loop (DL; see Fig. 6B). The 3′ end of the invading strand is used as the primer by DNA polymerases for DNA synthesis (postinvasion DNA synthesis), extending the DL (see Fig. 6F). After postinvasion DNA synthesis, repair may be completed by a break-induced replication, DSB repair, or synthesis-dependent strand-annealing pathway (5).Postinvasion DNA synthesis is crucial to high-fidelity repair of DSBs because it recovers the genetic information that might be lost during breakage events. Several DNA polymerases, including polymerase (Pol) δ, Pol η, and Pol ζ, are possibly involved in this process. Pol δ is known as a replicative polymerase that constitutes the replisome (17,...