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We investigated the relationship between oxidative stress and poor oocyte quality and whether the antioxidant melatonin improves oocyte quality. Follicular fluid was sampled at oocyte retrieval during in vitro fertilization and embryo transfer (IVF-ET). Intrafollicular concentrations of 8-hydroxy-2'-deoxyguanosine (8-OHdG) in women with high rates of degenerate oocytes were significantly higher than those with low rates of degenerate oocytes. As there was a negative correlation between intrafollicular concentrations of 8-OHdG and melatonin, 18 patients undergoing IVF-ET were given melatonin (3 mg/day), vitamin E (600 mg/day) or both melatonin and vitamin E. Intrafollicular concentrations of 8-OHdG and hexanoyl-lysine adduct were significantly reduced by these antioxidant treatments. One hundred and fifteen patients who failed to become pregnant with a low fertilization rate (< or =50%) in the previous IVF-ET cycle were divided into two groups during the next IVF-ET procedure; 56 patients with melatonin treatment (3 mg/day) and 59 patients without melatonin treatment. The fertilization rate was improved by melatonin treatment compared to the previous IVF-ET cycle. However, the fertilization rate was not significantly changed without melatonin treatment. Oocytes recovered from preovulatory follicles in mice were incubated with H2O2 for 12 hr. The percentage of mature oocytes with a first polar body was significantly reduced by addition of H2O2 (300 microm). The inhibitory effect of H2O2 was significantly blocked by simultaneous addition of melatonin. In conclusion, oxidative stress causes toxic effects on oocyte maturation and melatonin protects oocytes from oxidative stress. Melatonin is likely to improve oocyte quality and fertilization rates.

Severe acute respiratory syndrome (SARS) is caused by a coronavirus (SARS-CoV) and has the potential to threaten global public health and socioeconomic stability. Evidence of antibody-dependent enhancement (ADE) of SARS-CoV infection in vitro and in non-human primates clouds the prospects for a safe vaccine. Using antibodies from SARS patients, we identified and characterized SARS-CoV B-cell peptide epitopes with disparate functions. In rhesus macaques, the spike glycoprotein peptides S471-503, S604-625, and S1164-1191 elicited antibodies that efficiently prevented infection in non-human primates. In contrast, peptide S597-603 induced antibodies that enhanced infection both in vitro and in non-human primates by using an epitope sequence-dependent (ESD) mechanism. This peptide exhibited a high level of serological reactivity (64%), which resulted from the additive responses of two tandem epitopes (S597-603 and S604-625) and a long-term human B-cell memory response with antisera from convalescent SARS patients. Thus, peptide-based vaccines against SARS-CoV could be engineered to avoid ADE via elimination of the S597-603 epitope. We provide herein an alternative strategy to prepare a safe and effective vaccine for ADE of viral infection by identifying and eliminating epitope sequence-dependent enhancement of viral infection.

Flap endonuclease-1 (FEN1) is a key enzyme for maintaining genomic stability and replication. Proliferating cell nuclear antigen (PCNA) binds FEN1 and stimulates its endonuclease activity. The structural basis of the FEN1-PCNA interaction was revealed by the crystal structure of the complex between human FEN1 and PCNA. The main interface involves the C-terminal tail of FEN1, which forms two b-strands connected by a short helix, the bA-aA-bB motif, participating in b-b and hydrophobic interactions with PCNA. These interactions are similar to those previously observed for the p21 CIP1/WAF1 peptide. However, this structure involving the full-length enzyme has revealed additional interfaces that are involved in the core domain. The interactions at the interfaces maintain the enzyme in an inactive 'locked-down' orientation and might be utilized in rapid DNA-tracking by preserving the central hole of PCNA for sliding along the DNA. A hinge region present between the core domain and the C-terminal tail of FEN1 would play a role in switching the FEN1 orientation from an inactive to an active orientation.

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Proliferating cell nuclear antigen (PCNA) is essential for eukaryotic DNA replication and functions as a processivity factor of DNA polymerase ␦ (pol ␦). Due to the functional and structural similarity with the ␤-subunit of Escherichia coli DNA polymerase III, it has been proposed that PCNA would act as a molecular clamp during DNA synthesis. By site-directed mutagenesis and biochemical analyses, we have studied the functional domains of human PCNA required for stimulation of replication factor C (RF-C) ATPase and DNA synthesis by pol ␦. Short deletions from either the N or C termini caused drastic changes in extraction and chromatographic behaviors, suggesting that both of these terminal regions are crucial to fold the tertiary structure of PCNA. The short C-terminal stretch from Lys 254 to Glu 256 is necessary for stimulation of RF-C ATPase activity, but not for stimulation of DNA synthesis by pol ␦. Nine basic amino acids that are essential for activating DNA synthesis by pol ␦ are positioned at the internal ␣-helices of PCNA. This result is in good agreement with the observation that PCNA has a ring structure similar to the ␤-subunit and clamps a template DNA through this positively charged internal surface. Several other charged amino acids are also required to stimulate either RF-C ATPase or pol ␦ DNA synthesis. Some of them are positioned at loops which are exposed on one of the side surface of PCNA adjacent to the C-terminal loop. In addition, the ␤-sheets composing the intermolecular interface of the trimeric PCNA are important for interaction with pol ␦. Therefore, the outer surface of PCNA has multiple functional surfaces which are responsible for the interaction with multiple factors. Furthermore, the two side surfaces seem to be functionally distinguishable, and this may determine the orientation of tracking PCNA along the DNA.The proliferating cell nuclear antigen (PCNA) 1 is an essential replication factor for simian virus 40 (SV40) DNA replication in vitro and is involved in the elongation stages of DNA replication (1-4). Recent studies have further demonstrated the involvement of PCNA in cellular chromosomal DNA replication in vivo (5-7). In addition to its essential role in DNA replication, PCNA is required for nucleotide excision repair of DNA (8 -10) and also may participate in the cell cycle control as demonstrated by an interaction with a cyclin dependent kinase complex (11). Therefore, PCNA is multifunctional through the interaction with several specific partners, and all of the functions are crucial for cell proliferation.Genes encoding PCNA have been isolated from various eukaryotes and are composed of highly conserved amino acid sequences of around 260 residues. The functions of PCNA during DNA replication have been elucidated by studies of SV40 DNA replication in vitro. In this reaction, three protein components, PCNA, DNA polymerase (pol) ␦, and replication factor C (RF-C) are required for leading strand DNA synthesis following the initiation of DNA synthesis by DNA pol ␣ at the SV4...

Damaged DNA-binding protein, DDB, is a heterodimer of p127 and p48 with a high specificity for binding to several types of DNA damage. Mutations in the p48 gene that cause the loss of DDB activity were found in a subset of xeroderma pigmentosum complementation group E (XP-E) patients and have linked to the deficiency in global genomic repair of cyclobutane pyrimidine dimers (CPDs) in these cells. Here we show that with a highly defined system of purified repair factors, DDB can greatly stimulate the excision reaction reconstituted with XPA, RPA, XPC⅐HR23B, TFIIH, XPF⅐ERCC1 and XPG, up to 17-fold for CPDs and ϳ2-fold for (6-4) photoproducts (6-4PPs), indicating that no additional factor is required for the stimulation by DDB. Transfection of the p48 cDNA into an SV40-transformed human cell line, WI38VA13, was found to enhance DDB activity and the in vivo removal of CPDs and 6-4PPs. Furthermore, the combined technique of recently developed micropore UV irradiation and immunostaining revealed that p48 (probably in the form of DDB heterodimer) accumulates at locally damaged DNA sites immediately after UV irradiation, and this accumulation is also observed in XP-A and XP-C cells expressing exogenous p48. These results suggest that DDB can rapidly translocate to the damaged DNA sites independent of functional XPA and XPC proteins and directly enhance the excision reaction by core repair factors.Xeroderma pigmentosum (XP) 1 is a rare autosomal recessive disease characterized by sun sensitivity, pigmentation abnormalities, and high incidence of skin cancer (1, 2). XP is genetically heterogeneous and mutations in eight different genes (XPA through XPG and XPV) are known to cause this disease. All XP gene products, except XPV, are involved in nucleotide excision repair (NER), which removes a wide variety of DNA damages by dual incisions on both sides of the lesion (3-5). However, the function of damaged DNA-binding protein (DDB), which is linked to XP group E, is poorly understood.DDB was originally identified as a nuclear factor that binds to UV-damaged DNA and has been shown to recognize a wide spectrum of DNA lesions (6 -11). It is a heterodimer of p127 and p48, and both subunits are required for the activity (9, 12, 13). It has been reported that the mRNA levels of p48, but not of p127, strongly depend on the tumor suppressor p53 and increase further after DNA damage in a p53-dependent manner (14). Correspondingly, the protein levels and the activity of DDB also increase after UV irradiation (8,15). A subset of cell strains from XP group E patients were found to be deficient in the DDB activity (Ddb Ϫ XP-E) and to have mutations in p48 gene (7,12,16,17), although Ddb ϩ strains may have been misclassified as XP-E (18). The recent in vivo studies showed that XP-E cells are selectively defective in global genomic repair (GGR) (14), which repairs lesions from both nontranscribed genomic DNA and the nontranscribed strand of expressed genes. Chu and colleagues have demonstrated that Chinese hamster cells lack DDB activity...

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