A cell-based evaluation of human tyrosinase-mediated metabolic activation of leukoderma-inducing phenolic compounds.

BACKGROUND: Chemical leukoderma is a skin depigmentation disorder induced through contact with certain chemicals, most of which have a p-substituted phenol structure similar to the melanin precursor tyrosine. The tyrosinase-catalyzed oxidation of phenols to highly reactive o-quinone metabolites is a critical step in inducing leukoderma through the production of melanocyte-specific damage and immunological responses. OBJECTIVE: Our aim was to find an effective method to evaluate the formation of o-quinone by human tyrosinase and subsequent cellular reactions. METHODS: Human tyrosinase-expressing 293T cells were exposed to various phenolic compounds, after which the reactive o-quinones generated were identified as adducts of cellular thiols. We further examined whether the o-quinone formation induces reductions in cellular GSH or viability. RESULTS: Among the chemicals tested, all 7 leukoderma-inducing phenols/catechol (rhododendrol, raspberry ketone, monobenzone, 4-tert-butylphenol, 4-tert-butylcatechol, 4-S-cysteaminylphenol and p-cresol) were oxidized to o-quinone metabolites and were detected as adducts of cellular glutathione and cysteine, leading to cellular glutathione reduction, whereas 2-S-cysteaminylphenol and 4-n-butylresorcinol were not. In vitro analysis using a soluble variant of human tyrosinase revealed a similar substrate-specificity. Some leukoderma-inducing phenols exhibited tyrosinase-dependent cytotoxicity in this cell model and in B16BL6 melanoma cells where tyrosinase expression was effectively modulated by siRNA knockdown. CONCLUSION: We developed a cell-based metabolite analytical method to detect human tyrosinase-catalyzed formation of o-quinone from phenolic compounds by analyzing their thiol-adducts. The detailed analysis of each metabolite was superior in sensitivity and specificity compared to cytotoxicity assays for detecting known leukoderma-inducing phenols, providing an effective strategy for safety evaluation of chemicals.

Whole-genome analysis of human papillomavirus 67 isolated from Japanese women with cervical lesions.

BACKGROUND: Human papillomavirus (HPV) type 67 is phylogenetically classified into Alphapapillomavirus species 9 (alpha-9) together with other carcinogenic types (HPV16, 31, 33, 35, 52 and 58), but is the only alpha-9 type defined as possibly carcinogenic. This study aimed to determine whole-genome sequences of HPV67 isolated from Japanese women with cervical cancer or cervical intraepithelial neoplasia (CIN) to better understand the genetic basis of the oncogenic potential of HPV67. METHODS: Total cellular DNA isolated from cervical exfoliated cells that were single positive for HPV67 (invasive cervical cancer, n = 2; CIN3, n = 6; CIN 2, n = 1; CIN1, n = 2; the normal cervix, n = 1) was subjected to PCR to amplify HPV67 DNA, followed by next generation sequencing to determine the complete viral genome sequences. Variant lineages/sublineages were assigned through viral whole-genome phylogenetic analysis. The transcriptional activity of the virus early promoter was assessed by luciferase reporter assays in cervical cancer cell lines. RESULTS: Phylogenetic analyses of HPV67 genomes from Japan (n = 12) revealed that 11 belonged to lineage A (sublineage A1, n = 9; sublineage A2, n = 2) and one belonged to lineage B. All cancer cases contained sublineage A1 variants, and one of these contained an in-frame deletion in the E2 gene. The long control region of the HPV67 genome did not induce transcription from the virus early promoter in HeLa cells, but showed weak transcriptional activity in CaSki cells. CONCLUSIONS: The single detection of HPV67 in cervical cancer and precancer specimens strongly suggests the carcinogenic potential of this rare genotype. The phylogenetic analysis indicates a predominance of lineage A variants among HPV67 infections in Japan. Since only 23 complete genome sequences of HPV67 had been obtained until now, the newly determined genome sequences in this study are expected to contribute to further functional and evolutionary studies on the genetic diversity of HPV67.

The Oxidation of Equol by Tyrosinase Produces a Unique Di-ortho-Quinone: Possible Implications for Melanocyte Toxicity.

Equol (7-hydroxy-3-(4'-hydroxyphenyl)-chroman, EQ), one of the major intestinally derived metabolites of daidzein, the principal isoflavane found in soybeans and most soy foods, has recently attracted increased interest as a health-beneficial compound for estrogen-dependent diseases. However, based on its structure with two p-substituted phenols, this study aimed to examine whether EQ is a substrate for tyrosinase and whether it produces o-quinone metabolites that are highly cytotoxic to melanocyte. First, the tyrosinase-catalyzed oxidation of EQ was performed, which yielded three EQ-quinones. They were identified after being reduced to their corresponding catechols with NaBH4 or L-ascorbic acid. The binding of the EQ-quinones to N-acetyl-L-cysteine (NAC), glutathione (GSH), and bovine serum albumin via their cysteine residues was then examined. NAC and GSH afforded two mono-adducts and one di-adduct, which were identified by NMR and MS analysis. It was also found that EQ was oxidized to EQ-di-quinone in cells expressing human tyrosinase. Finally, it was confirmed that the EQ-oligomer, the EQ oxidation product, exerted potent pro-oxidant activity by oxidizing GSH to the oxidized GSSG and concomitantly producing H2O2. These results suggest that EQ-quinones could be cytotoxic to melanocytes due to their binding to cellular proteins.

Statins repress needle-like carbon nanotube- or cholesterol crystal-stimulated IL-1ß production by inhibiting the uptake of crystals by macrophages.

Statins are 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors that lower atherogenic LDL-cholesterol levels. Statins exert clinically relevant anti-inflammatory effects; however, the underlying molecular mechanism remains unclear. Studies have shown that endogenous and exogenous pathogenic crystals, such as cholesterol and monosodium urate (MSU), and needle-like nanomaterials, such as multi-wall carbon nanotubes (MWCNT), induce the production of IL-1ß and play a critical role in the development of crystal-associated sterile inflammatory pathologies. In this study, we evaluated the effect of statins on crystal-induced IL-1ß production in macrophages. We found that various statins, including pitavastatin, atorvastatin, fluvastatin, and lovastatin, but not squalene synthase inhibitor, repressed IL-1ß release upon MWCNT stimulation. In addition, IL-1ß production induced by cholesterol crystals and MSU crystals, but not by ATP or nigericin, was diminished. MWCNT-stimulated IL-1ß release was dependent on the expression of NLRP3, but not AIM2, NLRC4, or MEFV. Statin-induced repression was accompanied by reduced levels of mature caspase-1 and decreased uptake of MWCNT into cells. Supplementation of mevalonate, geranylgeranyl pyrophosphate, or farnesyl pyrophosphate prevented the reduction in IL-1ß release, suggesting a crucial role of protein prenylation, but not cholesterol synthesis. The statin-induced repression of MWCNT-elicited IL-1ß release was observed in THP-1-derived and mouse peritoneal macrophages, but not in bone marrow-derived macrophages where statins act in synergy with lipopolysaccharide to enhance the expression of IL-1ß precursor protein. In summary, we describe a novel anti-inflammatory mechanism through which statins repress mature IL-1ß release induced by pathogenic crystals and nanoneedles by inhibiting the internalization of crystals by macrophages.

Cas9 in Genetically Modified Food Is Unlikely to Cause Food Allergy.

Genome editing has undergone rapid development during the last three years. It is anticipated that genetically modified organisms (GMOs) for food purposes will be widely produced using the clustered regularly interspaced short palindromic repeat/Cas9 (CRISPR)/Cas9 system in the near future. However, the Cas9 gene may then enter the genomes of GMOs for food if the breeding process is not strictly managed, which could lead to the Cas9 protein or associated peptides being produced within these organisms. A variety of peptides could theoretically be produced from the Cas9 gene by using open reading frames different from that of Cas9 in the GMOs. In this study, Cas9 and the peptides potentially encoded by Cas9 genes were studied regarding their immunogenicity, in terms of the digestibility of Cas9 and the homology of the peptides to food allergens. First, the digestibility and thermal stability of Cas9 were studied. Digestibility was tested with natural or heat-denatured Cas9 in simulated gastric fluid in vitro. The two types of Cas9 were digested rapidly. Cas9 was also gradually degraded during heat treatment. Second, the peptides potentially encoded by Cas9 genes were examined for their homology to food allergens. Specifically, an 8-mer exact match search and a sliding 80-mer window search were performed using allergen databases. One of the peptides was found to have homology with a food allergen.

[A Patient with Recurrent Ovarian Cancer Who Was Hypersensitive to Carboplatin Exhibited Complete Response to Gemcitabine and Nedaplatin].

A 57-year-old multiparous woman with FIGO stage IV ovarian cancer underwent primary surgery and was administered postoperative chemotherapy consisting of paclitaxel and carboplatin (TC). Complete response was confirmed on computed tomography. After a 20-month platinum free interval (PFI), an elevated serum CA125 level and recurrence in the peritoneum were confirmed, and she was retreated with TC as second-line chemotherapy. A hypersensitivity reaction occurred after administering the second dose of carboplatin; therefore, carboplatin was changed to nedaplatin. Complete response was confirmed on computed tomography, and the serum CA125 level returned to normal. After an 8-month PFI, an elevated serum CA125 level and recurrence in the peritoneum and liver were confirmed, and she was treated with 6 cycles of combination chemotherapy consisting of gemcitabine (1,000 mg/m2: day 1 and 8 q3 weeks)and nedaplatin (80 mg/m2: day 1 q3 weeks). Only cytopenia (grade 2: CTCAE v4.0) was noted as a complication during chemotherapy. Complete response was confirmed on computed tomography. This report presents the case of a patient with recurrent ovarian cancer who was platinum sensitive and successfully treated with gemcitabine and nedaplatin after showing a hypersensitivity reaction to carboplatin.

Hypermutation in the E2 gene of human papillomavirus type 16 in cervical intraepithelial neoplasia.

Persistent infection with oncogenic human papillomavirus (HPV) causes cervical cancer. However, viral genetic changes during cervical carcinogenesis are not fully understood. Recent studies have revealed the presence of adenine/thymine-clustered hypermutation in the long control region of the HPV16 genome in cervical intraepithelial neoplasia (CIN) lesions, and suggested that apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (APOBEC) proteins, which play a key role in innate immunity against retroviral infection, potentially introduce such hypermutation. This study reports for the first time the detection of adenine/thymine-clustered hypermutation in the E2 gene of HPV16 isolated from clinical specimens with low- and high-grade CIN lesions (CIN1/3). Differential DNA denaturation PCR, which utilizes lower denaturation temperatures to selectively amplify adenine/thymine-rich DNA, identified clusters of adenine/thymine mutations in the E2 gene in 4 of 11 CIN1 (36.4%), and 6 of 27 CIN3 (22.2%) samples. Interestingly, the number of mutations per sample was higher in CIN3 than in CIN1. Although the relevance of E2 hypermutation in cervical carcinogenesis remains unclear, the observed hypermutation patterns strongly imply involvement of APOBEC3 proteins in editing the HPV16 genome during natural viral infection.

Human papillomavirus genotype distribution in cervical intraepithelial neoplasia grade 2/3 and invasive cervical cancer in Japanese women.

OBJECTIVE: Human papillomavirus vaccines are being introduced worldwide and are expected to reduce the incidence of cervical cancer. Here we report a cross-sectional study using a validated human papillomavirus genotyping method to reveal the human papillomavirus prevalence and genotype distribution in Japanese women with cervical intraepithelial neoplasia Grade 2/3 and invasive cervical cancer. METHODS: Cervical exfoliated cells were collected from 647 patients with abnormal cervical histology (cervical intraepithelial neoplasia Grade 2, n = 164; cervical intraepithelial neoplasia Grade 3, n = 334; and invasive cervical cancer, n = 149), and subjected to the PGMY-PCR-based genotyping assay. The association between human papillomavirus infection and lesion severity was calculated using a prevalence ratio. RESULTS: Overall, the prevalence of human papillomavirus deoxyribonucleic acid was 96.3% in cervical intraepithelial neoplasia Grade 2, 98.8% in cervical intraepithelial neoplasia Grade 3 and 88.0% in invasive cervical cancer (97.8% in squamous cell carcinoma and 71.4% in adenocarcinoma). The three most prevalent types were as follows: human papillomavirus 16 (29.3%), human papillomavirus 52 (27.4%) and human papillomavirus 58 (22.0%) in cervical intraepithelial neoplasia Grade 2; human papillomavirus 16 (44.9%), human papillomavirus 52 (26.0%) and human papillomavirus 58 (17.4%) in cervical intraepithelial neoplasia Grade 3; and human papillomavirus 16 (47.7%), human papillomavirus 18 (23.5%) and human papillomavirus 52 (8.7%) in invasive cervical cancer. The prevalence ratio of human papillomavirus 16 was significantly higher in cervical intraepithelial neoplasia Grade 3 compared with cervical intraepithelial neoplasia Grade 2 (prevalence ratio, 1.62; 95% confidence interval, 1.26-2.13) and in squamous cell carcinoma compared with cervical intraepithelial neoplasia Grade 3 (prevalence ratio, 1.55; 95% confidence interval, 1.25-1.87). Multiple infections decreased from cervical intraepithelial neoplasia Grade 2/3 (38.4/29.6%) to invasive cervical cancer (14.1%), whereas co-infections with human papillomavirus 16/52/58 were found in cervical intraepithelial neoplasia Grade 2/3. CONCLUSIONS: The results of this study provide pre-vaccination era baseline data on human papillomavirus type distribution in Japanese women and serve as a reliable basis for monitoring the future impact of human papillomavirus vaccination in Japan.

Genetic variation of human papillomavirus type 16 in individual clinical specimens revealed by deep sequencing.

Viral genetic diversity within infected cells or tissues, called viral quasispecies, has been mostly studied for RNA viruses, but has also been described among DNA viruses, including human papillomavirus type 16 (HPV16) present in cervical precancerous lesions. However, the extent of HPV genetic variation in cervical specimens, and its involvement in HPV-induced carcinogenesis, remains unclear. Here, we employ deep sequencing to comprehensively analyze genetic variation in the HPV16 genome isolated from individual clinical specimens. Through overlapping full-circle PCR, approximately 8-kb DNA fragments covering the whole HPV16 genome were amplified from HPV16-positive cervical exfoliated cells collected from patients with either low-grade squamous intraepithelial lesion (LSIL) or invasive cervical cancer (ICC). Deep sequencing of the amplified HPV16 DNA enabled de novo assembly of the full-length HPV16 genome sequence for each of 7 specimens (5 LSIL and 2 ICC samples). Subsequent alignment of read sequences to the assembled HPV16 sequence revealed that 2 LSILs and 1 ICC contained nucleotide variations within E6, E1 and the non-coding region between E5 and L2 with mutation frequencies of 0.60% to 5.42%. In transient replication assays, a novel E1 mutant found in ICC, E1 Q381E, showed reduced ability to support HPV16 origin-dependent replication. In addition, partially deleted E2 genes were detected in 1 LSIL sample in a mixed state with the intact E2 gene. Thus, the methods used in this study provide a fundamental framework for investigating the influence of HPV somatic genetic variation on cervical carcinogenesis.

Functional expression of amyloidogenic human stefins A and B in Pichia pastoris using codon optimization.

Complementary DNAs encoding human stefins A (HSA) and B (HSB) were synthesized using Pichia-preferred codons by overlap extension PCR. The full-length genes were ligated downstream of the glyceraldehyde-3-phosphate dehydrogenase promoter in the Pichia expression vector pGAPZαC and successfully expressed in Pichia pastoris strain X-33. Functional recombinant HSA and HSB proteins were purified from culture medium at yields of 121.3 ± 13.5 (n = 3) and 95.4 ± 4.1 (n = 3) mg/L, respectively. Using this expression strategy, we demonstrated that high levels of bioactive recombinant HSA and HSB can be produced by fermentation in P. pastoris.

Monoclonal antibodies recognizing cross-neutralization epitopes in human papillomavirus 16 minor capsid protein L2.

Antisera induced by immunization of rabbits with the synthetic peptide P56/75, which has the amino acid (aa) sequence from aa56 to aa75 of HPV16 L2, neutralize pseudovirions and raft-virions of multiple high-risk HPV types, indicating that cross-neutralization epitopes are present in the aa56-75 region. We generated two mouse monoclonal antibodies (MAb): MAb13B and MAb24B recognizing the regions of aa64-73 and aa58-64, respectively. The neutralization assay using pseudovirions of HPV16, 18, 31, 33, 35, 51, 52 and 58 showed that MAb13B neutralized HPV16, 18, and 51, and MAb24B neutralized all the types tested. The mixture of MAb13B and MAb24B neutralized HPV16, 18, and 51 pseudovirions more efficiently than each of the MAbs alone. The data indicate that there are at least two cross-neutralization epitopes in the aa56-75 region and that an antigen capable of presenting the two cross-neutralization epitopes would be a good vaccine candidate for a broad-spectrum of HPVs.

Do neutralizing antibody responses generated by human papillomavirus infections favor a better outcome of low-grade cervical lesions?

To determine the role of neutralizing antibody generated by human papillomavirus (HPV) infections, baseline levels of serum neutralizing antibodies directed against HPV 16 and cervical HPV DNA were determined in 242 unvaccinated women with low-grade cervical abnormalities, who were then monitored by cytology and colposcopy every 4 months. In women infected with HPV 16 (n = 42), abnormal cytology persisted longer in those positive for HPV 16-specific neutralizing antibodies at baseline (median time to cytological regression: 23.8 vs. 7.2 months). Progression to cervical precancer (cervical intraepithelial neoplasia grade 3) within 5 years occurred only among women carrying HPV 16-specific neutralizing antibodies (P = 0.03, log-rank test). In women infected with types other than HPV 16 (n = 200), detection of HPV 16-specific neutralizing antibodies was not correlated with disease outcome. In conclusion, development of specific neutralizing antibodies following natural HPV 16 infection did not favor a better outcome of low-grade cervical lesions induced by HPV 16 or by other types; rather, detection of neutralizing antibodies generated by current infection may reflect viral persistence and thus help identify those who are at high risk of disease progression.

Novel multiplexed genotyping of human papillomavirus using a VeraCode-allele-specific primer extension method.

A VeraCode-allele-specific primer extension (ASPE) method was applied to the detection and genotyping of human papillomavirus (HPV)-DNA. Oligonucleotide primers containing HPV-type-specific L1 sequences were annealed to HPV-DNA amplified by PGMY-PCR, followed by ASPE to label the DNA with biotinylated nucleotides. The labeled DNA was captured by VeraCode beads through hybridization, stained with a streptavidin-conjugated fluorophore, and detected by an Illumina BeadXpress® reader. By using this system, 16 clinically important HPV types (HPV6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68) were correctly genotyped in a multiplex format. The VeraCode-ASPE genotyping of clinical DNA samples yielded identical results with those obtained by validated PGMY-reverse blot hybridization assay, providing a new platform for high-throughput genotyping required for HPV epidemiological surveys.

Genotype distribution of human papillomaviruses in Japanese women with abnormal cervical cytology.

We report the prevalence and genotype distribution of human papillomaviruses (HPVs) among Japanese women with abnormal cervical cytology using the PGMY-CHUV assay, one of PGMY-PCR-based lineblot assays that was validated and shown to be suitable for the detection of multiple HPV types in a specimen with minimum bias. Total DNA was extracted from cervical exfoliated cells collected from 326 outpatients with abnormal Pap smears. Overall, 307 specimens (94%) were HPV-positive, 30% of which contained multiple genotypes. The prevalence of HPV DNA was 83% (49/59 samples) in atypical squamous cells of undetermined significance (ASC-US); 91% (20/22 samples) in atypical squamous cells, cannot exclude high-grade squamous intraepithelial lesion (ASC-H); 97% (130/134 samples) in low-grade squamous intraepithelial lesion (LSIL); and 99% (85/86 samples) in high-grade squamous intraepithelial lesion (HSIL). Three most frequent HPV types detected in HSIL were HPV16 (36%), HPV52 (24%), and HPV58 (14%). Our results suggest that multiple HPV infections are more prevalent in Japanese women than previously reported, and confirm that HPV52 and 58 are more dominant in their cervical precancerous lesions when compared to those reported in Western countries.

[A case of stage IVb vulvar cancer effectively treated by concurrent chemoradiotherapy with cisplatin].

A 72-year-old woman was hospitalized because of a 10 cm tumor in her right inguinal area. Furthermore, a 6 cm tumor mass was observed in her right vulva. Computed tomography revealed multiple swollen lymph nodes in the para-aortic and pelvic areas. On the basis of these findings, the patient was diagnosed with stage IVb squamous cell carcinoma of the vulva. Radiation therapy of 67.4 Gy/33 Fr was administered to the pelvis, inguinal area and vulva. Four courses of chemotherapy with cisplatin (40 mg/m(2)) were concurrently administered every week during radiation therapy. The response to chemoradiotherapy was assessed to be complete. The patient has been doing well without any recurrence for 24 months.

α-Synuclein aggregation and transmission are enhanced by leucine-rich repeat kinase 2 in human neuroblastoma SH-SY5Y cells.

Formation of α-synuclein aggregates is a key step in Parkinson's disease pathogenesis although the etiology remains elusive. α-Synuclein is accumulated in degenerating neurons, leading to the production of filamentous inclusions such as Lewy bodies. However, the in vitro overexpression of α-synuclein alone failed to induce inclusion bodies consisting of phosphorylated α-synuclein. The seeded aggregates-initiated polymerization of α-synuclein and tau has been reported elsewhere. What molecule is an initiator of filamentous inclusions remains to be defined. Here, we report that leucine-rich repeat kinase 2 (LRRK2)-cotransfection together with α-synuclein enhance the aggregate formation, phosphorylation, release to extracellular media of α-synuclein, and the cell-to-cell transmission into neighboring cells in human neuroblastoma SH-SY5Y cells. In cells transfected with α-synuclein alone, the proteins were distributed in the cytosol and did not form inclusions. On the other hand, the inclusions and phosphorylation of α-synuclein were formed in cells cotransfected with α-synuclein and LRRK2 G2019S mutant together. LRRK2 G2019S-cotransfected PC12 cells also induced the aggregates. Furthermore, the cell-to-cell transmission of α-synuclein and the cell toxicity were also enhanced by either LRRK2 wild type or G2019S mutant, whereas the cell viability was not decreased in cells transfected with α-synuclein alone. These results suggest that overexpression of LRRK2, especially G2019S mutant, whose functions remain unclear, initiate the aggregate formation, release and transmission of α-synuclein, resulting in the propagation of α-synuclein to neighboring cells and reduction of cell viability.

Biased amplification of human papillomavirus DNA in specimens containing multiple human papillomavirus types by PCR with consensus primers.

Genotyping human papillomavirus (HPV) in clinical specimens is important because each HPV type has different oncogenic potential. Amplification of HPV DNA by PCR with the consensus primers that are derived from the consensus sequences of the L1 gene has been used widely for the genotyping. As recent studies have shown that the cervical specimens often contain HPV of multiple types, it is necessary to confirm whether the PCR with the consensus primers amplifies multiple types of HPV DNA without bias. We amplified HPV DNA in the test samples by PCR with three commonly used consensus primer pairs (L1C1/L1C2+C2M, MY09/11, and GP5+/6+), and the resultant amplicons were identified by hybridization with type-specific probes on a nylon membrane. L1C1/L1C2+C2M showed a higher sensitivity than the other primers, as defined by the ability to detect HPV DNA, on test samples containing serially diluted one of HPV16, 18, 51, 52, and 58 plasmids. L1C1/L1C2+C2M failed to amplify HPV16 in the mixed test samples containing HPV16, and either 18 or 51. The three consensus primers frequently caused incorrect genotyping in the selected clinical specimens containing HPV16 and one or two of HPV18, 31, 51, 52, and 58. The data indicate that PCR with consensus primers is not suitable for genotyping HPV in specimens containing multiple HPV types, and suggest that the genotyping data obtained by such a method should be carefully interpreted.

Constituents of cultivated Agaricus blazei.

Two phenylhexane derivatives (1, 2), benzoylergostane (3), N-benzoyl-L-leucine methyl ester (4), two known ergostanes, and highly degraded incisterol were isolated from fruit bodies of Agaricus blazei. Compound 3 exhibited strong cytotoxicity toward HepG2 cells (IC(50) = 6.0 ± 0.33 µM).

Inhibition of nuclear entry of HPV16 pseudovirus-packaged DNA by an anti-HPV16 L2 neutralizing antibody.

Rabbit anti-HPV16 L2 serum (anti-P56/75) neutralizes multiple oncogenic human papillomaviruses (HPVs). We inoculated HeLa cells with HPV16 pseudovirus (16PV) and with anti-P56/75-bound 16PV (16PV-Ab). Both 16PV and 16PV-Ab attached equally well to the cell surface. However, the cell-attached L1 protein of 16PV became trypsin-resistant after incubation at 37°C, whereas approximately 20% of the cell-attached 16PV-Ab L1 remained trypsin-sensitive. Confocal microscopy of HeLa cells inoculated with 16PV revealed packaged DNA in the nucleus at 22h after inoculation; however, nuclear DNA was not detected in cells inoculated with 16PV-Ab. Electron microscopy of HeLa cells inoculated with 16PV showed particles located in multivesicular bodies, lamellar bodies, and the cytosol after 4h; no cytosolic particles were detected after inoculation with 16PV-Ab. These data suggest that anti-P56/75 inhibits HPV infection partly by blocking viral entry and primarily by blocking the transport of the viral genome to the nucleus.

Poly(ADP-ribose) polymerase (PARP)-1-independent apoptosis-inducing factor (AIF) release and cell death are induced by eleostearic acid and blocked by alpha-tocopherol and MEK inhibition.

Poly(ADP-ribose)polymerase-1 (PARP-1) is thought to be required for apoptosis-inducing factor (AIF) release from mitochondria in caspase-independent apoptosis. The mechanism by which AIF is released through PARP-1 remains unclear. Here, we provide evidence that PARP-1-independent AIF release and cell death are induced by a trienoic fatty acid, alpha-eleostearic acid (alpha-ESA). Alpha-ESA induced the caspase-independent and AIF-initiated apoptotic death of neuronal cell lines, independently of PARP-1 activation. The cell death was inhibited by the MEK inhibitor U0126 and by knockdown of MEK using small interfering RNA. However, inhibitors for JNK, p38 inhibitors, calpain, phospholipase A(2), and phosphatidylinositol 3-kinase, did not block cell death. AIF was translocated to the nucleus after the induction of apoptosis by alpha-ESA in differentiated PC12 cells without activating caspase-3 and PARP-1. The alpha-ESA-mediated cell death was not inhibited by PARP inhibitor 3,4-dihydro-5-[4-(1-piperidinyl)butoxyl]-1(2H)-isoquinoline and by knockdown of PARP-1 using small interfering RNA. Unlike N-methyl-N'-nitro-N-nitrosoguanidine treatment, histone-phosphorylated histone 2AX was not phosphorylated by alpha-ESA, which suggests no DNA damage. Overexpression of Bcl-2 did not inhibit the cell death. alpha-ESA caused a small quantity of superoxide production in the mitochondria, resulting in the reduction of mitochondrial membrane potential, both of which were blocked by a trace amount of alpha-tocopherol localized in the mitochondria. Our results demonstrate that alpha-ESA induces PARP-1-independent AIF release and cell death without activating Bax, cytochrome c, and caspase-3. MEK is also a key molecule, although the link between ERK, AIF release, and cell death remains unknown. Finding molecules that regulate AIF release may be an important therapeutic target for the treatment of neuronal injury.