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A coiled-coil microtubule-bundling protein, p180, was originally identified as one of the ribosome receptor candidates on the rough endoplasmic reticulum (ER) and is highly expressed in secretory tissues. Recently, we reported that p180 plays crucial roles in upregulating collagen biosynthesis, mainly by facilitating ribosome association on the ER. Here, we provide evidence that p180 is required to form translationally active polysome/translocon complexes on the ER. Assembly of highly-developed polysomes on the ER was severely perturbed upon loss of p180. p180 associates with polysome/translocon complexes through multiple contact sites: it was coimmunoprecipitated with the translocon complex independently of ribosomes, while it can also bind to ribosomal large subunit specifically. The responsible domain of p180 for membrane polysome assembly was identified in the C-terminal coiled-coil region. The degree of ribosome occupation of collagen and fibronectin mRNAs was regulated in response to increased traffic demands. This effect appears to be exerted in a manner specific for a specified set of mRNAs. Collectively, our data suggest that p180 is required to form translationally active polysome/translocon complexes on the ER membrane, and plays a pivotal role in highly efficient biosynthesis on the ER membrane through facilitating polysome formation in professional secretory cells.

The ability of mammalian cytidine deaminases encoded by the APOBEC3 (A3) genes to restrict a broad number of endogenous retroelements and exogenous retroviruses, including murine leukemia virus and human immunodeficiency virus (HIV)-1, is now well established. The RNA editing family member apolipoprotein B (apo B)-editing catalytic subunit 1 (APOBEC1; A1) from a variety of mammalian species, a protein involved in lipid transport and which mediates C–U deamination of mRNA for apo B, has also been shown to modify a range of exogenous retroviruses, but its activity against endogenous retroelements remains unclear. Here, we show in cell culture-based retrotransposition assays that A1 family proteins from multiple mammalian species can also reduce the mobility and infectivity potential of LINE-1 (long interspersed nucleotide sequence-1, L1) and long-terminal repeats (LTRs) retrotransposons (or endogenous retroviruses), such as murine intracisternal A-particle (IAP) and MusD sequences. The anti-L1 activity of A1 was mainly mediated by a deamination-independent mechanism, and was not affected by subcellular localization of the proteins. In contrast, the inhibition of LTR-retrotransposons appeared to require the deaminase activity of A1 proteins. Thus, the AID/APOBEC family proteins including A1s employ multiple mechanisms to regulate the mobility of autonomous retrotransposons in several mammalian species.

BACKGROUND: Hemagglutination-inhibition (HI) antibody titers correlate with protective immunity to seasonal influenza viruses. However, inactivated H5N1 influenza vaccines from Vietnam 2004 strains afford protection without producing high or even detectable HI antibodies. METHODS: BALB/c mice were immunized twice (at a 3-week interval) with inactivated whole-virus influenza vaccine produced using reverse genetics, with the internal genes of A/PR/8/34 (a high-yield strain) and the hemagglutinin (HA) and neuraminidase (NA) genes of A/Vietnam/1194/04 (H5N1) virus (NIBRG-14) adjuvanted with alum (5 microg of HA). Either HA- or NA-binding antibodies were absorbed from the immune serum. The protective efficacy of these antibodies was determined by injecting the absorbed serum into severe combined immunodeficiency mice, which were then challenged with highly pathogenic H5N1 virus (A/Vietnam/Jp1203/2004; Japanese isolate of A/Vietnam/1203/2004). RESULTS: The NIBRG-14 vaccine elicited levels of anti-HA antibodies similar to levels elicited by the H1N1 vaccines, as well as levels of anti-NA antibodies higher than those elicited by the H1N1 vaccines. The absorption of either anti-HA or anti-NA antibody from immune serum samples obtained from NIBRG-14-vaccinated mice significantly reduced the protective efficacy of the serum. CONCLUSIONS: For NIBRG-14 vaccines to confer protection to vaccinated hosts, both anti-HA and anti-NA antibodies are required. This finding implies that the measurement of both antibody levels may be required for accurate evaluation of vaccine efficacy.

Approximately 17% of the human genome is comprised of long interspersed nuclear element 1 (LINE-1, L1) non-LTR retrotransposons. L1 retrotransposition is known to be the cause of several genetic diseases, such as hemophilia A, Duchene muscular dystrophy, and so on. The L1 retroelements are also able to cause colon cancer, suggesting that L1 transposition could occur not only in germ cells, but also in somatic cells if innate immunity would not function appropriately. The mechanisms of L1 transposition restriction in the normal cells, however, are not fully defined. We here show that antiretroviral innate proteins, human APOBEC3 (hA3) family members, from hA3A to hA3H, differentially reduce the level of L1 retrotransposition that does not correlate either with antiviral activity against Vif-deficient HIV-1 and murine leukemia virus, or with patterns of subcellular localization. Importantly, hA3G protein inhibits L1 retrotransposition, in striking contrast to the recent reports. Inhibitory effect of hA3 family members on L1 transposition might not be due to deaminase activity, but due to novel mechanism(s). Thus, we conclude that all hA3 proteins act to differentially suppress uncontrolled transposition of L1 elements.

Background Accurate numbers of copies of Kaposi sarcoma-associated herpesvirus (KSHV) and numbers of virus-infected cells in lesions caused by KSHV-associated diseases are unknown. Methods Quantitative polymerase chain reaction (PCR) and computerized imaging of immunohistochemical analysis were performed on pathologic sections of samples from persons with KSHV-associated diseases. Results Real-time PCR and semiquantitative PCR-Southern blotting demonstrated that DNA extracted from biopsy samples of KS lesions contained approximately 1-2 viral copies/cell. KSHV-associated lymphoma contained 10-50 viral copies/cell. Computerized-image analysis demonstrated that approximately 49% of cells expressed KSHV-encoded latency-associated nuclear antigen in KS biopsy samples. On the basis of results of real-time PCR and computerized-image analysis, the predicted number of viral copies was 3.2 viral copies/cell in KS lesions. Computerized-image analysis also revealed that the expression of open-reading frame (ORF)-50 protein, an immediate early protein of KSHV, was very rare in KS lesions, which implies that they were mainly composed of proliferating cells latently infected with KSHV. In multicentric Castleman disease lesions, 25% of virus-infected cells expressed ORF50 protein, which suggests the frequent lytic replication of KSHV. Conclusions Numbers of viral copies and of virus-positive cells vary among KSHV-associated diseases, which suggests different mechanisms of viral pathogenesis. The combination of real-time PCR and computerized-image analysis provides a useful tool for the assessment of the number of viral copies in KSHV-associated diseases.

Background. Now that highly active antiretroviral therapy (HAART) is being initiated on a large scale in West Africa, it remains controversial whether protease inhibitors (Pis), originally designed and tested against human immunodeficiency virus type 1 (HIV-1) subtype B, are equally effective against the non-B subtypes that are prevalent in West African countries. In this study, we investigated whether Ghanaian HIV-1 isolates, as representatives of West African isolates, are susceptible to PIs. Methods. We first generated an HIV-1 protease cassette vector proviral DNA carrying a luciferase gene, which allows patient-derived HIV-1 proteases to be inserted and to be subjected to both genotypic and phenotypic assays. HIV-1 protease genes derived from 39 treatment-naive Ghanaian patients were used in this experiment as representatives of West African strains. The cloned patient-derived HIV-1 protease genes were first sequenced and then genetically compared. Phenotypic analysis was performed with Ghanaian HIV-1 protease-chimeric viruses in the presence of 6 different PIs. Structural models of HIV-1 protease homodimers were constructed by the molecular modeling software. Results. Genetic analysis of cloned patient-derived HIV-1 protease genes indicated that most of the Ghanaian HIV-1 proteases are placed as subtype CRF02_AG strains, which are phylogenetically distant from subtype B strains, and that Ghanaian HIV-1 proteases do not harbor known major mutations influencing drug resistance but commonly carry 2-3 minor mutations. Phenotypic analysis performed with HIV-1 protease-recombinant viruses in the presence of 6 different PIs revealed that Ghanaian HIV-1 proteases are differentially less susceptible to the PIs. In support of this finding of differential susceptibility, structural analysis showed a significant distortion ofnelfinavir, but not of amprenavir, in the Ghanaian protease pocket, suggesting nelfinavir might be less insertable into the Ghanaian protease than into the protease of subtype B. Conclusions. These findings provide implications for the combination of Pis during the introduction of HAART into West Africa.

Background Epidermodysplasia verruciformis (EV) is a rare skin disease characterized by disseminated pityriasis versicolor-like or flat wart-like lesions and by the development of skin carcinomas. It is well established that specific cutaneous human papillomaviruses (EV–HPVs) are associated with both benign and malignant skin lesions in EV patients. However, little is known of the relationship between HPV and the mucosal lesions of EV patients. Objectives To detect and identify HPV types associated with skin and mucosal lesions of an EV patient. Patient/methods We investigated the skin carcinoma and the coexisting tonsillar carcinoma of a 41-year-old man with EV. Histopathologically, both lesions were squamous cell carcinomas. We analysed these two lesions by immunohistochemistry, in situ hybridization, and by molecular virology. Results Neither skin nor tonsillar lesions exhibited positivity for HPV capsid antigen by immunohistochemistry. By Southern blot hybridization, however, the skin carcinoma harboured ‘EV-specific’ HPV20 DNA, while the tonsillar carcinoma harboured ‘genital’ HPV16 DNA. In addition, in situ hybridization localized the respective viral DNA in the corresponding lesion. Conclusions The results indicate that EV–HPV could be responsible for the development of the skin carcinoma, but not the mucosal carcinoma in this patient.

Glycosyltransferases can exhibit tissue-specific expression. By histochemistry glycosyltransferases and their products can be localized to specific cell types in organs of complex cellular composition. We have applied the lectin Amaranthin, having a nominal specificity for Galbeta1,3GalNAcR and Neu5Ac2,3Galbeta1, 3GalNAcalpha-R, and a monoclonal antibody raised against Galbeta1, 3GalNAcalphaR to examine the distribution of these simple O-glycans in adult rat kidney. The monoclonal antibody stained ascending thin limbs of Henle, distal convoluted tubules, and collecting ducts of cortex and outer medulla. Remarkably, the ascending thick limb of Henle, located between ascending thin limb and distal convoluted tubules, was unreactive. However, Amaranthin staining was detectable in ascending thick limbs of Henle, in addition to the structures positive with the monoclonal antibody. In kidney extracts, two bands of approximately 160 kDa and >210 kDa were reactive with both Amaranthin and the monoclonal antibody. One band at approximately 200 kDa, and a smear at approximately 100 kDa, were reactive only with Amaranthin. Our data show that in rat kidney simple O-linked glycans are expressed in a highly specialized manner along the renal tubule and can be detected only on a few glycoproteins. This may reflect a cell-type-specific expression of the corresponding glycosyltransferases.

A woman with Hailey-Hailey disease, suffering from carcinoma of the vulva, was examined by histology and for the presence of human papillomavirus (HPV) DNA by polymerase chain reaction (PCR) and in situ hybridization. Our diagnosis by histological examination revealed the vulval carcinoma to be a squamous cell carcinoma (SCC), adjacent to lesions of Hailey-Hailey disease and severe dysplasia/carcinoma in situ [vulval intraepithelial neoplasia (VIN) III]. The PCR with consensus primers for the L1 region (L1-PCR) successfully amplified HPV DNA using total DNA extracted from formalin-fixed and paraffin-embedded tissue specimens. Restriction fragment length polymorphism analysis and sequencing of L1-PCR products revealed HPV types 16 and 39. HPV 16-specific primers for the E6 region identified HPV 16 DNA. In situ hybridization analysis with biotinylated HPV 16 and 39 DNA probes revealed the presence of the HPV 39 genome in the nuclei of the tumour cells in the SCC. These results indicate that HPV 16 and 39 are associated with lesions in vulval carcinoma. Regarding the patient's susceptibility to infection in the case of Hailey-Hailey disease, there is a possibility that HPV was inoculated into the lesions of Hailey-Hailey disease and induced those of VIN III and SCC.

Samples of lung tissue, taken at autopsy, from 10 Japanese patients with primary pulmonary hypertension (PPH) and samples of lung tissue from 12 Japanese patients with secondary pulmonary hypertension were tested for the presence of human herpesvirus 8 (HHV-8). All samples from patients with PPH contained plexiform lesions around pulmonary arterial vessels, but immunohistochemistry failed to detect the HHV-8-encoded latency-associated nuclear antigen. HHV-8 DNA could not be amplified by polymerase chain reaction for the HHV-8-encoded K1 and KS330(233) genes in any sample. These data suggest that HHV-8 infection is not associated with PPH in Japanese patients.

Human papillomavirus (HPV) can be detected in skin lesions of Bowen's disease, particularly on the fingers, and its genotype is associated with mucosal/genital types of HPV. We report herein an 85-year-old woman who had HPV-associated Bowen's disease on her elbow. HPV-58 DNA was detected in the lesion by polymerase chain reaction with restriction fragment length polymorphism and by Southern blot hybridization. In situ hybridization revealed numerous hybrid cells in the nuclei of the upper epidermis and stratum corneum of Bowen's disease. A high-risk type of mucosal HPV-58 DNA is associated with Bowen's disease in this case, suggesting that HPV-related Bowen's disease is not always restricted to genital or finger lesions.