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A commercial optical wireless LAN system has a base station attached to a ceiling, and a number of LEDs therein broadcast optical beams to movable stations below. The movable stations always communicate with the base station when they are in a service area. The LEDs are arranged so that their combined optical beams effectively irradiate the service area. However, studies concerning such designs have not been reported and thus LEDs in base stations have been arranged empirically. The minimum optical intensity in the service area is a key parameter in designing systems because it determines the optical power margin of the system and should be increased as much as possible. We examined the optimum arrangement of LEDs with which the minimum optical intensity in the service area is maximized, assuming the total number and power of LEDs are fixed. Referring to the commercial systems, we assumed LEDs are aligned on coaxial circles in a rotationally symmetric scheme and thus a disk-shaped service area is implemented. We assumed LEDs have the same beam profile, but each group aligned on a different circle has its own number of elements and inclination angle with respect to the vertical axis. We compared numerical results with our experimental results. This study will contribute to designing the base stations of optical wireless LANs.

We give a new version of W. L. Edge’s construction of the linear system of plane sextics containing Wiman’s sextic, by means of configuration space of 5 points on projective line. This construction reveals out more of the inner beauty of the hidden geometry of Wiman’s sextic. Furthermore, it allows one to give a friendly proof for the fact that the linear system is actually a pencil, the fact that is important in both Edge’s and our constructions.

Infection of endothelial cells with human herpesvirus 8 (HHV-8) is an essential event in the development of Kaposi's sarcoma. When primary microvascular endothelial cells (MECs) were infected with HHV-8 at a low multiplicity of infection, considerable latent replication of HHV-8 occurred, leading to a time-dependent increase in the percentage of virus-infected cells that was accompanied by cellular spindling and growth to a high density with loss of contact inhibition. Only a low percentage of MECs supported lytic replication of HHV-8 and produced infectious virus. Phosphonoformic acid blocked production of infectious virus but did not inhibit the rapid expansion of latently infected MECs. Pretreatment of MECs with alpha interferon (IFN-α) prior to infection effectively reduced HHV-8 viral gene expression, latent replication, and production of infectious virus. High levels of the double-stranded RNA activated protein kinase (PKR) were expressed in HHV-8-infected cells, and incubation with IFN-α increased PKR expression more in virus-infected cells than in uninfected cells. MECs that were immortalized with simian virus 40 large-T antigen differed from nonimmortalized MECs in their response to infection with HHV-8 and demonstrated that cells with elevated levels of expression of antiviral transcripts expressed viral transcripts at reduced levels. These studies demonstrate that MECs respond to HHV-8 with enhanced expression of cellular antiviral genes and that augmentation of innate antiviral defenses with IFN-α is a more effective strategy than inhibition of viral lytic replication to protect MECs from infection with HHV-8 and to restrict proliferation of virus-infected MECs.

Copyright © American Society for Microbiology, Journal of Virology, volume 77, 8147-8152, 2003 publisher, To analyze the mechanisms of entry of human herpesvirus 8 (HHV-8), we established a reporter cell line T1H6 that contains the lacZ gene under the control of the polyadenylated nuclear RNA promoter, known to be strongly activated by a viral transactivator, Rta. We found that infection with cell-free virus, as well as cocultivation with HHV-8-positive primary effusion lymphoma cell lines, activated the lacZ gene of T1H6 in a sensitive and dose-dependent manner. Addition of Polybrene and centrifugation enhanced, but polysulfonate compounds inhibited, the HHV-8 infectivity. RGD-motif-containing polypeptides and integrins did not decrease the infectivity, suggesting the presence of an additional cellular receptor other than the reported one. The entry was dependent on pH acidification but not on the clathrin pathway. Although conditioned media obtained from human immunodeficiency virus (HIV)-infected cells did not have any effect on the early steps of HHV-8 infection, intracellular expression of a proviral HIV type 1, but not of Tat alone, increased the HHV-8-dependent reporter activation slightly, suggesting a potential of HIV-mediated enhancement of an early step of HHV-8 infection.

Plasma levels of prolyl-hydroxyproline (Pro-Hyp) and hydroxyprolyl-glycine (Hyp-Gly) in healthy volunteers (n=5) after ingestion of collagen hydrolysate were estimated by liquid chromatography-tandem mass spectrometry. The ratio of Hyp-Gly to Pro-Hyp was distributed in the range of 0.063-0.221. This is a first report for quantification of food-derived Hyp-Gly in human plasma.

We have observed the oscillation of a waveguide mode free‐electron laser using the coherent synchrotron radiation emitted from electron bunches. The oscillation was observed at 2.73 mm with a linewidth of 0.4%. This spectral selection was attributed to the dispersion of the radiation in the waveguide. We found that the oscillating radiation had to have the same group velocity as the velocity of the electrons passing through the wiggler field.

The key regulator of the switch from latent to lytic replication of the human herpesvirus 8 (HHV-8; KSHV) is the replication and transcription activator (Rta). The ability of Rta to regulate cellular gene expression was examined by transient transfection into cells that were not infected with HHV-8. Rta induced some, but not all, NF-kappa B-responsive reporters through mechanisms that did not involve activation of classic forms of NF-kappa B. Furthermore, transfection of the NF-kappa B subunit Rel A inhibited the ability of Rta to transactivate some but not all reporters. For example, Rel A inhibited the ability of Rta to transactivate the IL-6 promoter, but only when sequences upstream of the NF-kappa B site were present. The ability of Rel A to inhibit Rta-mediated transactivation was not dependent on a functional NF-kappa B site within the promoter, suggesting an indirect mechanism for inhibition. These studies suggest that Rta expression during lytic reactivation of HHV-8 would lead to expression of some cellular genes, including IL-6, whereas activation of NF-kappa B could inhibit some responses to Rta.

Adjuvant arthritic rats are known to be more susceptible to gastric damage induced by non-steroidal anti-inflammatory drugs (NSAIDs) than are normal rats. We compared the relative gastric safety profile of etodolac with those of meloxicam, diclofenac sodium and indometacin in adjuvant arthritic rats and normal rats or mice. As a measure of the safety profiles of NSAIDs, we used the safety index, the ratio of the dose that elicits gastric mucosal lesions to the effective dose as an anti-inflammatory or analgesic compound. The anti-inflammatory or analgesic effects of NSAIDs were assessed by paw swelling in adjuvant arthritic rats, and either carrageenin-induced paw edema or brewer’s yeast-induced hyperalgesia, as well as acetic acid-induced writhing, in normal rats or mice. In addition, we also investigated the effects of these NSAIDs on human COX-1 and COX-2 activity. Etodolac and other NSAIDs inhibited paw swelling and caused gastric mucosal lesions in adjuvant arthritic rats in a dose-dependent manner. Etodolac showed the highest UD50 value and safety index among these NSAIDs in arthritic rats. In normal rats, etodolac also showed the highest UD50 value and safety index, except when its effects were assessed by acetic acid-induced writhing. Etodolac and meloxicam showed selectivity for human COX-2 over COX-1. In contrast, diclofenac sodium and indometacin were selective for COX-1. These results suggest that etodolac, a COX-2 selective NSAID, has anti-inflammatory effects with a better safety profile for the stomach than do non-selective NSAIDs, including diclofenac sodium and indometacin, in adjuvant arthritic as well as normal rats.

Introduction: Engineering an antigen receptor gene in patients' lymphocytes is one promising strategy to create antigen-specific lymphocytes without senescent phenotypes. The strategy provides an opportunity to extend the application of adoptive T cell therapy for cancer patients. However, this concept has not been tested in epithelial cancer patients. Material and methods: MAGE-A4-specific TCR α and β chains were isolated from a human T cell clone that recognizes MAGE-A4 143-151 peptide in an HLA-A*24:02 restricted manner. This T cell clone did not show any cross reactivity to the peptides with a homology to the MAGE-A4 epitope. A retroviral vector that encodes these TCR chains without any artificial modification was constructed; the lymphocytes transduced with the retroviral vector killed the MAGE-A4 expressing tumor in vitro and inhibited the tumor growth in the NOG immmunodeficient mice. Patients were eligible if they had previously-treated recurrent MAGE-A4-expressing esophageal cancer, and were positive for HLA-A*24;02. Lymphocytes harvested from the patients were infected with the retroviral vector. The TCR-gene transduced T lymphocytes were once transferred to the patients without lymphocyte-depleting treatment, and MAGE-A4 peptide was given 2 and 4 weeks after. The cell doses were divided into 3 cohorts of 2x108 1x109 and 5x109, with a dose-escalating design, by evaluating the safety. Results: 10 patients received the TCR-gene transduced T lymphocytes. No adverse events related to the cell transfer were observed. The TCR-gene transduced lymphocytes were detected in their peripheral blood in all 10 patients, which showed a dose-dependent appearance during the first 14 days, reaching peak and plateau levels from 3 to 7 days, and declined within 14 days. The cells persisted at 0.5% to 1% level in the peripheral mononuclear cells from day 14 to 63 after the cell transfer. In 6 patients whose blood samples had been collected for over 6 months, 3 patients maintained stable levels as long as 16 months, maintaining the immune reactivity to MAGE-A4-expressing tumor cells. In one patient, whose esophageal tumor was biopsied after the transfer, the TCR-gene transduced cells were detected in the tumor site. 7 patients developed tumor progressions within 2 months after the transfer. Their overall survivals were ranged from 3 to 18+ months, with a median of 10. 3 patients who had minimal tumor lesions at baseline have been free from disease-progression for 12, 15, and 19 months, respectively. Conclusion: Wild-type TCR-gene transduced lymphocytes targeting MAGE-A4 antigen were safely given to refractory esophageal cancer patients. The cells persisted in their peripheral blood in a dose-dependent manner in the early phase, and they have been stably persisting over 6 months. Three patients are free from disease progression more than a year. These results encourage us to proceed to further phase trials. Citation Format: Shinichi Kageyama, Hiroaki Ikeda, Naoko Imai, Mikiya Ishihara, Yoshihiro Miyahara, Shugo Ueda, Takeshi Ishikawa, Hiroaki Naota, Kohshi Ohishi, Taizo Shiraishi, Naoki Inoue, Masashige Tanabe, Tomohide Kidokoro, Hirofumi Yoshioka, Daisuke Tomura, Ikuei Nukaya, Junichi Mineno, Kazutoh Takesako, Naoyuki Katayama, Hiroshi Shiku. Adoptive transfer of wild-type TCR gene transduced T lymphocytes targeting MAGE-A4 antigen to patients with refractory esophageal cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr CT212. doi:10.1158/1538-7445.AM2014-CT212

Improvement of serologic assays for detection of antibodies against human herpesvirus 8 (HHV-8) is critical to better understand its epidemiology and biology. We produced the HHV-8 latent (ORF73) and lytic (ORF65, K8.1, and glycoprotein B) antigens in the Semliki Forest virus system and evaluated their performance in immunofluorescence assays (IFAs) and enzyme-linked immunosorbent assays (ELISAs). These assays were compared with other latent antigen-based assays, including an IFA based on primary effusion lymphoma (PEL) cells and an ELISA based on bacterially expressed ORF73 antigen, as well as with other lytic antigen-based assays, including an IFA based on induced PEL cells, a commercial ELISA based on purified virions, and ELISAs based on K8.1- and ORF65-derived oligopeptides. We used a panel of 180 serum specimens obtained from three groups expected to have high, intermediate, and low HHV-8 prevalences. Using three different evaluation methods, we found that (i) the performances of the lytic antigen-based ELISAs were almost equivalent, (ii) the lytic antigen-based assays were more sensitive than the latent antigen-based assays, and (iii) in general, IFAs were more sensitive than ELISAs based on the same open reading frame. We also found that serum specimens from healthy individuals contained antibodies cross-reactive with HHV-8 glycoprotein B that can potentially cause false-positive reactions in lytic PEL-based IFAs. Although this is not a substantial problem in most epidemiologic studies, it may confound the interpretation of data in studies that require high assay specificity. Because the K8.1-based IFA provides sensitivity similar to that of lytic PEL-based IFAs and improved specificity, it can be a useful alternative to the PEL-based IFAs.

Several assays have been developed for detection of immunoglobulin G antibodies to Human herpesvirus 8 (HHV-8), including immunofluorescence assays (IFAs) and enzyme-linked immunosorbent assays (ELISAs). However, the specificity and sensitivity of these assays are not completely defined due to the lack of a “gold standard.” Although IFAs based on primary effusion lymphoma (PEL) cell lines are used widely, the assays can be confounded by nonspecific reactions against cellular components and potential cross-reaction with antibodies against other herpesviruses. To provide more reliable IFAs, we established recombinant Semliki Forest viruses (rSFVs) expressing the HHV-8-specific proteins ORF73 and K8.1 and used BHK-21 cells infected with these rSFVs for IFA (ORF73-IFA and K8.1-IFA). Expression of the HHV-8-specific proteins at very high levels by the rSFV system allowed easy scoring for IFA and thereby increased specificity. The rSFV system also allowed detection of antibodies against glycosylation-dependent epitopes of K8.1. Titers measured by rSFV-based IFAs and PEL-based IFAs correlated well (correlation coefficients of >0.9), and concordances of seroreactivities between rSFV-based and PEL-based IFAs were >97% (κ > 0.93). K8.1-IFA was more sensitive than either ORF73-IFA or peptide ELISAs. Using PEL-based lytic IFA as a reference assay, the sensitivity and specificity of K8.1-IFA were estimated to be 94 and 100%, respectively. HHV-8 prevalences determined by K8.1-IFA among the human immunodeficiency virus (HIV)-positive (HIV + ) Kaposi's sarcoma (KS) patients, HIV + KS − patients, and healthy controls were 100, 65, and 6.7%, respectively, which were consistent with prior reports. Therefore, our rSFV-based IFAs may provide a specific and sensitive method for use in epidemiology studies. In addition, they will provide a basis for further development of diagnostic tests for HHV-8 infection.

To the Editor: Varicella-zoster virus (VZV), a human herpesvirus, is the causative agent of varicella (chickenpox) and herpes zoster (shingles). Worldwide, children are routinely vaccinated with a live attenuated varicella vaccine containing the Oka vaccine (vOka) strain of VZV, originally developed in Japan (1–3). Although the risk for secondary transmission of the vOka strain from immunocompromised vaccinees to susceptible persons is relatively high, the risk for transmission from immunocompetent vaccinees is low (1). We report secondary transmission of the vOka strain from an immunocompetent girl with a history of varicella vaccination to her healthy susceptible brother. Herpes zoster developed in a healthy 3-year-old girl 2 years after she had received the varicella vaccine (lot VZ040; Biken, Osaka, Japan). She received oral acyclovir treatment and fully recovered by day 19 after herpes zoster onset. On the same day that the girl recovered, her immunocompetent 2-year-old brother was found to have fever and a rash consisting of 10–20 papulovesicles; mild varicella was diagnosed. The boy had no known history of contact with persons infected with varicella or with persons who administered the varicella vaccine. After receiving oral acyclovir treatment, the boy recovered without systemic complications. On day 19 after the girl’s onset of herpes zoster, an enzyme immunoassay (Denka Seiken, Tokyo, Japan) confirmed the presence of VZV-specific immunoglobulin (Ig) G (titer 48.9, well above the detection limit of 2.0) but not IgM. The boy showed seroconversion of VZV-specific IgG from a titer of

Gastrointestinal Endoscopy, 69(1), pp.158-159; 2009

Human herpesvirus 6 variants A and B (HHV-6A and HHV-6B) are closely related viruses that can be readily distinguished by comparison of restriction endonuclease profiles and nucleotide sequences. The viruses are similar with respect to genomic and genetic organization, and their genomes cross-hybridize extensively, but they differ in biological and epidemiologic features. Differences include infectivity of T-cell lines, patterns of reactivity with monoclonal antibodies, and disease associations. Here we report the complete genome sequence of HHV-6B strain Z29 [HHV-6B(Z29)], describe its genetic content, and present an analysis of the relationships between HHV-6A and HHV-6B. As sequenced, the HHV-6B(Z29) genome is 162,114 bp long and is composed of a 144,528-bp unique segment (U) bracketed by 8,793-bp direct repeats (DR). The genomic sequence allows prediction of a total of 119 unique open reading frames (ORFs), 9 of which are present only in HHV-6B. Splicing is predicted in 11 genes, resulting in the 119 ORFs composing 97 unique genes. The overall nucleotide sequence identity between HHV-6A and HHV-6B is 90%. The most divergent regions are DR and the right end of U, spanning ORFs U86 to U100. These regions have 85 and 72% nucleotide sequence identity, respectively. The amino acid sequences of 13 of the 17 ORFs at the right end of U differ by more than 10%, with the notable exception of U94, the adeno-associated virus type 2 rep homolog, which differs by only 2.4%. This region also includes putative cis -acting sequences that are likely to be involved in transcriptional regulation of the major immediate-early locus. The catalog of variant-specific genetic differences resulting from our comparison of the genome sequences adds support to previous data indicating that HHV-6A and HHV-6B are distinct herpesvirus species.

Synovial tissue affected by rheumatoid arthritis is characterized by proliferation, which leads to irreversible cartilage and bone destruction. Current and experimental treatments have been aimed mainly at correcting the underlying immune abnormalities, but these treatments often prove ineffective in preventing the invasive destruction. We studied the expression of cyclin-dependent kinase inhibitors in rheumatoid synovial cells as a means of suppressing synovial cell proliferation. Synovial cells derived from hypertrophic synovial tissue readily expressed p16INK4a when they were growth-inhibited. This was not seen in other fibroblasts, including those derived from normal and osteoarthritis-affected synovial tissues. In vivo adenoviral gene therapy with the p16INK4a gene efficiently inhibited the pathology in an animal model of rheumatoid arthritis. Thus, the induction of p16INK4a may provide a new approach to the effective treatment of rheumatoid arthritis.

Although vectors based on adeno-associated virus (AAV) offer several unique advantages, their usage has been hampered by the difficulties encountered in vector production. In this report, we describe a new AAV packaging system based on inducible amplification of integrated helper and vector constructs containing the simian virus 40 (SV40) replication origin. The packaging and producer cell lines developed express SV40 T antigen under the control of the reverse tetracycline transactivator system, which allows inducible amplification of chromosomal loci linked to the SV40 origin. Culturing these cells in the presence of doxycycline followed by adenovirus infection resulted in helper and vector gene amplification as well as higher vector titers. Clonal producer cell lines generated vector titers that were 10 times higher than those obtained by standard methods, with approximately 10 4 vector particles produced per cell. These stocks were free of detectable replication-competent virus. The lack of a transfection step combined with the reproducibility of stable producer lines makes this packaging method ideally suited for the large-scale production of vector stocks for human gene therapy.

HHV-6 genes and their functions that are unique and different from those of HCMV are summarized. HHV-6 encodes a HSV-1 UL9 homolog that is an origin-binding protein (OBP) essential for HSV-1 DNA replication, although HCMV has no clear homolog. The HHV-6 OBP binds to sequences with similarity to alpha-herpesvirus replication origin that lie within a genomic segment serving as an origin in transient assays. Consensus sequence for OBP binding, protein domain analyses and a model for OBP-origin interactions are described. HHV-6 encodes an AAV-2 rep homolog. Its transcript is expressed at low mRNA levels as a spliced transcript. The gene product suppresses both transformation by H.ras and transcription by HIV-1 promoters. HHV-6 specific transactivator gene DR7 exhibits malignant transforming activity and its protein binds to p53. Studies on IE-A locus and an HCMV UL69 homolog are also discussed.

The phenomena in short pulsed laser irradiation to a pure aluminum substrate are analyzed on the basis of thermohydrodynamics to evaluate the mechanism of bump formation in laser texturing for a magnetic disk. The analysis model is made for calculation of surface profile, temperature field and velocity field assuming that the vaporization recoil pressure, surface tension and thermocapillay force (Marangoni effect) act at the free surface with taking account of the evaporated mass loss. It is found that a crater shape bump is formed by the flow induced by the vaporization recoil pressure mainly and that thermocapillary force makes a bump higher while outside of bump-formed area starts to solidify.The phenomena in short pulsed laser irradiation to a pure aluminum substrate are analyzed on the basis of thermohydrodynamics to evaluate the mechanism of bump formation in laser texturing for a magnetic disk. The analysis model is made for calculation of surface profile, temperature field and velocity field assuming that the vaporization recoil pressure, surface tension and thermocapillay force (Marangoni effect) act at the free surface with taking account of the evaporated mass loss. It is found that a crater shape bump is formed by the flow induced by the vaporization recoil pressure mainly and that thermocapillary force makes a bump higher while outside of bump-formed area starts to solidify.

Some experiments of laser texturing for magnetic memory disks were carried out in order to estimate the effect of evaporation to bump formation. Considering the experimental results, a mathematical model based on thermohydrodynamics was established to analyze fusion phenomena accompanied with evaporation. Latent heat of vaporization, movement of gas and liquid interface accompanied with evaporation, evaporation recoil force and surface tension caused by temperature gradient were considered. VOF method was applied to numerical computations of the free surface problem. Some numerical examples were calculated and bump formation process was discussed. As the results, it was made clear that quantity of evaporation is a little, but surface geometry of molten metal is influenced by movement of gas and liquid interface accompanied with evaporation greatly in addition to effect of flow of liquid metal in the molten pool.Some experiments of laser texturing for magnetic memory disks were carried out in order to estimate the effect of evaporation to bump formation. Considering the experimental results, a mathematical model based on thermohydrodynamics was established to analyze fusion phenomena accompanied with evaporation. Latent heat of vaporization, movement of gas and liquid interface accompanied with evaporation, evaporation recoil force and surface tension caused by temperature gradient were considered. VOF method was applied to numerical computations of the free surface problem. Some numerical examples were calculated and bump formation process was discussed. As the results, it was made clear that quantity of evaporation is a little, but surface geometry of molten metal is influenced by movement of gas and liquid interface accompanied with evaporation greatly in addition to effect of flow of liquid metal in the molten pool.

Human herpesvirus 7 (HHV-7) is a close relative of human herpesvirus 6A (HHV-6A) and human herpesvirus 6B (HHV-6B) based on limited biologic and genetic data. In this work we describe physical and genetic maps for HHV-7 strain SB [HHV-7(SB)], which was obtained from the saliva of a healthy adult. The HHV-7(SB) genome length is approximately 144 kb by clamped homogeneous electric field gel electrophoresis and approximately 135 kb by summation of restriction endonuclease fragments. We constructed plasmid clones and PCR amplimers that span the HHV-7 genome, except for the genomic termini, and determined the maps of the restriction endonuclease cleavage sites for BamHI, PstI, and SacI. The HHV-7(SB) genome is composed of a single unique region of approximately 122 kb bounded at each end by a 6 kb direct repeat. Homologs to thirty-five herpesvirus genes were identified. The highest similarity was with the HHV-6 genes, with an average amino acid identity of 50%, followed by the human cytomegalovirus counterpart. The genomic and genetic maps indicated that the HHV-7 and HHV-6 genomes are colinear. There was no sequence variation in a segment of the gene encoding the DNA polymerase-associated factor homolog among six HHV-7 isolates, while the corresponding segment of the HHV-6A and HHV-6B counterparts differed by 4.6%. These data support previous observations that the closest genetic relatives of HHV-7 are betaherpesviruses.

Human herpesvirus 6(HHV-6) variants A and B differ in cell tropism, reactivity with monoclonal antibodies, restriction endonuclease profiles, and epidemiology. Nonetheless, comparative nucleotide and amino acid sequences from several genes indicate that the viruses are very highly conserved genetically, The B variant is the major etiologic agent of exanthem subitum and is frequently isolated from children with febrile illness; no disease has been etiologically associated with HHV-6A. One HHV-6A strain has been cloned and sequenced, but similar information and reagents are not available for HHV-6B. We report here the determination of maps of the restriction endonuclease cleavage sites for BamHI, C1aI, HindIII, KpnI, and Sa1I, and the cloning in plasmids and bacteriophages of fragments representing over 95% of the HHV-6B strain Z29 [HHV-6B(Z29)] genome. Hybridization experiments and orientation of several blocks of nucleotide sequence information onto the genomic map indicate that HHV-6A and HHV-6B genomes are colinear.

We previously demonstrated by a DNA-binding assay that the human herpesvirus 6B (HHV-6B) replication origin has a structure similar to those of alphaherpesviruses, although the HHV-6B and herpes simplex virus type 1 (HSV-1) origin-binding proteins (OBPs) and origins are not interchangeable. Here we describe additional properties of the interaction between HHV-6B OBP and the HHV-6B origin. Competitive electrophoretic mobility shift assays (EMSAs) with DNA duplexes containing single-base alterations allowed deduction of a consensus DNA sequence for HHV-6B-specific OBP binding, YGWYCWCCY, where Y is T or C and W is T or A, while that for HSV-1-specific binding was reported to be YGYTCGCACT. By EMSA, the HHV-6B OBP DNA-binding domain was mapped to a segment containing amino acids 482 to 770. However, in Southwestern (protein-DNA) blotting, the region sufficient for the DNA binding encompassed only amino acids 657 to 770. Similarly, Southwestern blotting showed that amino acids 689 to 851 of HSV-1 OBP had HSV-1 origin-binding activity, although this region was insufficient for origin binding in the EMSA. Although the longer DNA-binding domains identified by EMSA have marginal overall homology among HHV-6B and alphaherpesvirus OBP homologs, the smaller regions sufficient for the binding observed by Southwestern blotting have significant similarity. From these results, we propose a hypothesis that the DNA-binding domain of herpesvirus OBPs consists of two subdomains, one containing a conserved motif that contacts DNA directly, and another, less well conserved, that may modulate either the conformation or accessibility of the binding domain.

Congenital cytomegalovirus (cCMV) infection occurs in 0.2–2% of all births in developed countries and causes developmental abnormalities.1 In addition to patients symptomatic at birth, asymptomatic newborns can develop late-onset sequelae, including sensorineural hearing loss and developmental delay. As the early identification of congenitally infected newborns may allow early intervention and antiviral treatment options, it is important to establish newborn cCMV screening programmes. Since newborn screening assays using dried blood spots for cCMV infection were …

We obtained isolates of human herpesvirus 7 (HHV-7) from 6 of 8 healthy adults by culturing saliva with human umbilical cord blood lymphocytes. These isolates were identified as HHV-7 on the basis of comparisons of restriction endonuclease fragment profiles and hybridization with HHV-7 strain RK DNA. The isolates could be differentiated from HHV-7 strain RK and from each other by their restriction endonuclease fragment profiles. We confirm the finding of frequent isolation of HHV-7 from saliva of healthy adults and report the first dual isolation of human herpesvirus 6 (HHV-6) and HHV-7 from a single saliva specimen. We also describe an in situ hybridization assay that can distinguish between HHV-6 and HHV-7.

High-energy x-ray and neutron diffraction measurements on polycrystalline La(2/3-x)Li(3x)TiO(3) (0.075x0.165) were performed. The total scattering structure factors were analysed by the reverse Monte Carlo (RMC) modelling technique, resulting in three-dimensional particle configurations. These configurations were then used for revealing the distributions of La and Li ions and to understand the relationship between these distributions and ionic conduction. An alternating arrangement of La-rich and La-poor layers along the c-axis was found in the x = 0.075 composition. Intriguingly, this arrangement has gradually disappeared in samples with higher Li concentration. Furthermore, RMC models exhibit disordered distributions of Li ions, situated mainly on the La-rich layer, and there is a significant probability of Li ions occupying the interstitial sites (T site) between the O-3 triangle plane of the TiO(6) octahedron and an La ion or its vacancy site. It was also found on the basis of the RMC models that the bond valence sum (BVS) for Li ions behaves differently on La-rich and La-poor layers at low Li concentration compositions, but they are similar at high Li concentration compositions. This is consistent with the behaviour of the alternating arrangement of La-rich and La-poor layers. It is also suggested that the Li ions around the bottleneck at (1/2, 0, 0) (bottom layer) can jump to an adjacent bottleneck at (0, 1/2, 0) through the T site and not only Li ions in the La-poor layers but also Li ions in the La-rich layers contribute to the bottleneck-bottleneck Li conduction.

To examine if Epstein-Barr virus (EBV) infection is associated with the two autoimmune diseases, Sjogren's syndrome and rheumatoid arthritis, IgG antibody titers of sera from patients with the disorders were evaluated for five constituents of EBV nuclear antigens (EBNA-1, -2, -3, -4, and -6). To circumvent interference by autoantibodies in the sera, fusion proteins synthesized in Escherichia coli were used as specific antigens. By ELISA the average IgG antibody titers to domains of the five EBNAs, especially the amino-terminal domain of EBNA-2, in sera of patients with Sjogren's syndrome were slightly higher than those in normal sera. The tendency of sera from Sjogren's syndrome patients to have higher reactivities to the EBNA domains was also observed by immunoblotting. By comparison, few heightened serologic responses to EBNAs were observed in sera from patients with rheumatoid arthritis. Further analyses are required to determine if EBV is associated with Sjogren's syndrome in any way.

The domain of Epstein-Barr virus nuclear antigen 1 (EBNA-1) which is essential for binding to a region containing oriP , an episomal replication origin of EBV DNA, was analyzed by DNA binding assay with β-galactosidase-EBNA-1 fusion proteins. It was revealed that a 159-amino acid (aa) domain, 460–618 aa, of EBNA-1 retained the oriP -binding activity and the domain's activity was abolished by a deletion of 29 as from its amino-terminal end and by a 38 aa deletion from its carboxyl-end as well. One of five monoclonal antibodies against EBNA-1 specifically inhibited the binding of the β-galactosidase-EBNA-1 fusion protein to the oriP region. The epitope recognized by the monoclonal antibody was mapped in the crucial 29 as region. An analysis of the domain's putative secondary structure and a computer search of amino acid sequence homology indicated that the 159-aa domain contains the hypothetical basic-helix-loop-helix structure which is considered to be a common characteristic structure of a family of DNA binding proteins. Examinations of DNA binding activity of the other EBNA polypeptides with a series of fusion proteins and similar structural analyses of their amino acid sequences were also performed. This study suggests that EBNA-1 is a constituent of the family of DNA binding proteins which are involved in transcriptional regulation critical for cell differentiation or cell-type determination.

By S1 nuclease protection mapping, we characterized RNA transcripts and nascent ColE1 DNA synthesized in wild-type Escherichia coli cells after infection with lambda-mini-ColE1 hybrid bacteriophages. Transcription of the RNA II region of ColE1 DNA in vivo starts mostly from the RNA II promoter, which was identified by in vitro experiments, and ends at or near the ori site. Synthesis of the leading strand of ColE1 DNA was found to start at the ori site. Nevertheless, the molar ratio of the nascent DNA to the synthesized transcripts ending at the ori site was less than 0.05. In bacterial rnh mutants whose RNase H activities were less than 0.06% of that of the wild type, transcription patterns, as well as nascent DNA synthesis, were still similar to those in rnh+ cells. However, in bacteria whose rnh gene was interrupted by insertion of a drug resistance gene, the number of transcripts ending at the ori site was much reduced and that of transcripts reading through the ori site was definitely increased relative to that observed in wild-type bacteria. These results suggested that cleavage of the RNA transcript at the ori site in vivo is dependent on RNase H activity, as demonstrated in the in vitro system, but most of the cleaved RNA is unable to prime initiation of ColE1 DNA synthesis efficiently.

The driving force for formation of a surface relief grating on an azobenzene-containing polymer is theoretically analyzed and experimentally confirmed by polarization analyses. We formulate the driving force based on the Coulomb interaction between an optical electric field and its induced polarization charges. The formula derived includes not only the optically induced gradient force but also the force due to the spatial modulation of the refractive index. The latter stems from the reorientation of the azobenzene molecules in the polymer through the cyclic trans-cis photoisomerization caused by the Ar+ laser used to construct the interference pattern.

Metastatic, chemotherapy-resistant melanoma is an intractable cancer with a very poor prognosis. As to immunotherapy targeting metastatic melanoma, HLA-A2+ patients were mainly enrolled in the study in Western countries. However, HLA-A24+ melanoma patients-oriented immunotherapy has not been fully investigated. In the present study, we investigated the effect of dendritic cell (DC)-based immunotherapy on metastatic melanoma patients with HLA-A2 or A24 genotype. Nine cases of metastatic melanoma were enrolled into a phase I study of monocyte-derived dendritic cell (DC)-based immunotherapy. HLA-genotype analysis revealed 4 cases of HLA-A*0201, 1 of A*0206 and 4 of A*2402. Enriched monocytes were obtained using OptiPrep™ from leukapheresis products, and then incubated with GM-CSF and IL-4 in a closed serum-free system. After pulsing with a cocktail of 5 melanoma-associated synthetic peptides (gp100, tyrosinase, MAGE-2, MAGE-3 and MART-1 or MAGE-1) restricted to HLA-A2 or A24 and KLH, cells were cryopreserved until used. Finally, thawed DCs were washed and injected subcutaneously (s.c.) into the inguinal region in a dose-escalation manner. The mean percentage of DCs rated as lin-HLA-DR+ in melanoma patients was 46.4 ± 15.6 %. Most of DCs expressed high level of co-stimulatory molecules and type1 phenotype (CD11c+HLA-DR+), while a moderate number of mature DCs with CD83 and CCR7 positive were contained in DC products. DC injections were well tolerated except for transient liver dysfunction (elevation of transaminases, Grade I-II). All 6 evaluable cases except for early PD showed positive immunological responses to more than 2 melanoma peptides in an ELISPOT assay. Two representative responders demonstrated strong HLA-class I protein expression in the tumor and very high scores of ELISPOT that might correlate to the regression of metastatic tumors. Clinical response through DC injections was as follows : 1CR, 1 PR, 1SD and 6 PD. All 59 DC injections in the phase I study were tolerable in terms of safety, however, the maximal tolerable dose of DCs was not determined. These results suggested that peptide cocktail-treated DC-based immunotherapy had the potential for utilizing as one of therapeutic tools against metastatic melanoma in Japan.