A Comparison of the Pharmacokinetics and Drug Safety Among East Asian Populations.

Global clinical studies conducted in various countries and regions are increasing. Race and extrinsic ethnic factors are key covariates that may affect the pharmacokinetics (PK), efficacy, and safety of the drug. Genetic similarity among East Asian populations has been confirmed; thus, PK, efficacy, and safety in these populations are expected to be similar, but this has not been confirmed. This study presents a comparison of PK and safety among East Asians from clinical studies sponsored by Pfizer. Four compounds with different characteristics, including mechanism of actions and PK profiles, were selected, and retrospective PK and safety comparisons in East Asians were conducted. No distinct differences were observed in PK and safety across the 4 compounds. These results are consistent with previous reports on PK comparisons and meet the expectations based on genetic similarity among East Asians. Extrapolation of these findings to other compounds should be done with caution, but these results should support the consideration of mutual use of clinical data among East Asian countries.

Protective effect of cepharanthin on cisplatin-induced renal toxicity through metallothionein expression.

AIMS: Cisplatin (CDDP) is a potent anticancer agent, but severe renal toxicity can limit its use. We investigated the protective effect of cepharanthin (CE), a biscoclaurin alkaloid, on the renal toxicity of CDDP. MAIN METHODS: Mice were given CDDP along with CE. Effects of CE on CDDP toxicity were investigated by assaying markers of renal toxicity together with MT expression, and by histopathological examination of the kidney. MT-null mice were also examined. KEY FINDINGS: CE induced expression of metallothionein (MT). Pre-administration of CE attenuated an increase in blood urea nitrogen (BUN) concentrations after the CDDP injection. A histochemical analysis demonstrated protection against CDDP-induced necrocytosis of kidney tissues by CE. The protective effect of CE did not occur in the MT-null mice. SIGNIFICANCE: Pretreatment with CE may reduce the renal toxicity of CDDP through expression of MT.

Generalized myositis mimicking polymyositis associated with chronic active Epstein-Barr virus infection.

BACKGROUND: Chronic generalized myositis has not so far been reported as a complication of chronic active Epstein-Barr virus infection (CAEBV). We encountered three patients with chronic generalized myositis mimicking polymyositis associated with CAEBV. METHODS: To clarify the pathological character of this myositis, we investigated the distribution, clonality, and the immunophenotype of EBV-infected cells and lymphocytes infiltrating in muscles. RESULTS: Clinically, two patients showed symmetrical proximal weakness and muscle atrophy as the initial and main symptom. Although the condition resembled polymyositis, they had also lingual and/or orbital myositis. The other patient showed generalized myositis at the late phase of CAEBV. In all of them, immunotherapy was ineffective and prognosis was poor. Intramuscular infiltrating lymphocytes in our patients were mainly CD45RO+, CD3+, CD4-, CD8-, TCR betaF1-, TCR deltaTCS1-, CD56-, CD79a-, CD21-, HLA-DR+, ZEBRA -, LMP1-, and EBER+ T cells. Oligoclonal expansion of EBV-infected T cells was shown in the muscles. However, there were no malignant lymphocytes. CONCLUSIONS: This new form of myositis must be distinguished from polymyositis and the other conventional forms of myositis. Careful investigation of hidden CAEBV is recommended when patients present with steroid non-responsive chronic progressive generalized myositis, in particular, with lingual or orbital involvement.

Epstein-Barr virus (EBV) nuclear antigen leader protein (EBNA-LP) forms complexes with a cellular anti-apoptosis protein Bcl-2 or its EBV counterpart BHRF1 through HS1-associated protein X-1.

Epstein-Barr virus (EBV) nuclear antigen leader protein (EBNA-LP) plays a critical role in EBV-induced transformation. An earlier report (Y. Kawaguchi et al., J. Virol. 74: 10104-10111, 2000) showed that EBNA-LP interacts with a cellular protein HS1-associated protein X-1 (HAX-1). The predicted amino acid sequence of HAX-1 exhibits similarity to that of another cellular protein Nip3 which has been shown to interact with cellular and viral anti-apoptotic proteins such as Bcl-2 and BHRF1, an EBV homolog of Bcl-2. Here we investigated whether HAX-1, like Nip3, interacts with Bcl-2 proteins and report the following. (i) A purified chimeric protein consisting of gluthathione S-transferase (GST) fused to BHRF1 (GST-BHRF1) or Bcl-2 (GST-Bcl-2) specifically pulled down HAX-1 transiently expressed in COS-7 cells. (ii) GST-BHRF1 or GST-Bcl-2 was not able to pull down EBNA-LP transiently expressed in COS-7 cells, whereas each of the GST fusion proteins formed complexes with EBNA-LP in the presence of RAX-1. These results indicated that EBNA-LP interacts with the viral and cellular Bcl-2 proteins through HAX-1, suggesting that EBNA-LP possesses a potential function in the regulation of apoptosis in EBV-infected cells.

Physical interaction of Epstein-Barr virus (EBV) nuclear antigen leader protein (EBNA-LP) with human oestrogen-related receptor 1 (hERR1): hERR1 interacts with a conserved domain of EBNA-LP that is critical for EBV-induced B-cell immortalization.

Epstein-Barr virus (EBV) nuclear antigen leader protein (EBNA-LP) consists of W1W2 repeats and a unique C-terminal Y1Y2 domain and plays a critical role in EBV-induced transformation. To identify the cellular proteins associating with EBNA-LP, we performed a yeast two-hybrid screen using EBNA-LP cDNA containing a single W1W2 domain as bait and an EBV-transformed human peripheral blood lymphocyte cDNA library as the source of cellular genes. Our results were as follows. (i) A cDNA in the positive yeast colony was found to encode a cellular protein, human oestrogen-related receptor 1 (hERR1), which is a constitutive transcriptional activator of the various types of oestrogen response elements. (ii) A purified chimeric protein consisting of glutathione S-transferase (GST) fused to hERR1 specifically formed complexes with EBNA-LPs containing one (EBNA-LPR1), two (EBNA-LPR2) or four W1W2 repeats (EBNA-LPR4) transiently expressed in COS-7 cells. Reciprocally, GST fused to EBNA-LPR1 or EBNA-LPR2 pulled down hERR1 transiently expressed in COS-7 cells. (iii) Mutational analyses of EBNA-LP revealed that the Y2 domain of EBNA-LP is responsible for the interaction with hERR1 and two leucines in the Y2 domain (Leu-78 and -82), which are conserved among a subset of primate gammaherpesviruses, are interactive sites for hERR1. So far, it has been reported that the only domain of EBNA-LP critical for EBV-induced transformation is the Y1Y2 domain. Potential roles of hERR1 in EBV-induced transformation are discussed.

Novel immediate-early protein IE19 of human cytomegalovirus activates the origin recognition complex I promoter in a cooperative manner with IE72.

The major immediate-early (MIE) gene of human cytomegalovirus (HCMV) expresses IE86, IE72, IE55, and IE18 mRNA by differential splicing. Reverse transcription-PCR with IE72-specific primers generated an 0.65-kb cDNA from HCMV-infected fibroblast RNA, which does not correspond to any known MIE cDNA. Nucleotide sequencing revealed that the 0.65-kb cDNA is from exons 1, 2, and 3 and part of exon 4, indicating that it is derived from a novel alternatively spliced mRNA of the MIE gene. The cDNA encodes a 172-amino-acid polypeptide, termed IE19, which corresponds to an IE72 variant with an internal deletion from Val(86) to Pro(404) and appears as a band at 38 kDa on a sodium dodecyl sulfate-polyacrylamide gel. IE19 mRNA was expressed at a low level in the immediate-early, early, and late period of viral infection. IE19 was localized in nuclei, and a transient-expression assay revealed that IE19 enhances IE72-dependent activation of the HsOrc1 promoter, which is identified here as an IE72 target promoter. Another MIE protein, IE86, activated the same promoter but only weakly compared to IE72, and coexpression of IE19 did not alter the IE86-mediated transcriptional activation. In addition, IE19 did not enhance the IE72-dependent activation of the HCMV UL54 promoter. These results suggest that IE19 is a transcriptional coactivator that works with IE72.

Identification and characterization of Marek's disease virus serotype 1 (MDV1) ICP22 gene product: MDV1 ICP22 transactivates the MDV1 ICP27 promoter synergistically with MDV1 ICP4.

A previous report [Virus Genes 6 (1992) 365-378] has shown that the US1 gene of Marek's disease virus serotype 1 (MDV1) encodes a homologue of herpes simplex virus type 1 infected cell protein No. 22 (ICP22). In the present study, we expressed and identified a product of the MDV1 US1 gene in chicken embryo fibroblasts (CEFs) with the aid of a recombinant baculovirus expressing a Flag epitope-tagged MDV1 US1 gene, under control of the SRalpha promoter (composed of the enhancer region of the simian virus 40 early promoter and the R region of the human T-cell leukaemia virus type 1 long terminal repeat). In CEF infected with the recombinant baculovirus, MDV1 ICP22 was specifically and efficiently expressed in the presence of n-butyric acid. The apparent M(r) of the expressed protein was 30,000. Reporter gene assays revealed that MDV1 ICP22 by itself transactivated an MDV1 ICP27 promoter/reporter construct weakly but specifically, and furthermore, worked synergistically with MDV1 ICP4 to efficiently up-regulate the MDV1 ICP27 promoter. MDV1 ICP22 may be a regulatory protein that stimulates viral promoters in co-operation with other viral regulatory proteins such as MDV1 ICP4.

Conserved region CR2 of Epstein-Barr virus nuclear antigen leader protein is a multifunctional domain that mediates self-association as well as nuclear localization and nuclear matrix association.

Self-association of viral proteins is important for many of their functions, including enzymatic, transcriptional, and transformational activities. Epstein-Barr virus (EBV) nuclear antigen leader protein (EBNA-LP) contains various numbers of W1W2 repeats and a unique carboxyl-terminal Y1Y2 domain. It was reported that EBNA-LP associates with a variety of cellular proteins and plays a critical role in EBV-induced transformation. We report here that EBNA-LP self-associates in vivo and the domain responsible for the homotypic association is a multifunctional domain mediating nuclear localization, nuclear matrix association, and EBNA-2-dependent coactivator function of the protein. Our conclusions are based on the following observations. (i) EBNA-LP interacts with itself or its derivatives in the yeast two-hybrid system. (ii) A purified chimeric protein consisting of glutathione S-transferase fused to EBNA-LP specifically formed complexes with EBNA-LP transiently expressed in COS-7 cells. (iii) When Flag epitope-tagged EBNA-LP with either one or two W1W2 repeats and EBNA-LP containing four W1W2 repeats were coexpressed in COS-7 cells, the latter was specifically coimmunoprecipitated with the former. (iv) Mutational analyses of EBNA-LP with deletion mutants revealed that the region between codons 19 and 39 (relative to the first amino acid residue of the W2 domain) is essential for self-association of the protein. The mapped region almost completely overlaps with CR2 and CR3, regions conserved among a subset of primate gamma-herpesviruses and critical for EBNA-2-dependent coactivator function. Amino acid substitutions in CR2 alone abolished the ability of the protein to self-interact. This laboratory previously reported that CR2 is also responsible for nuclear localization and nuclear matrix association (A. Yokoyama, Y. Kawaguchi, I. Kitabayashi, M. Ohki, and K. Hirai, Virology 279:401-413, 2001). (v) Sucrose gradient sedimentation showed that amino acid substitutions in CR2 reduced the ability of the protein to form protein complexes in B cells. These results suggest that self-association of EBNA-LP may be important for its various functions and interactions of the protein with multiple cellular proteins.

Epstein-Barr virus-encoded protein kinase BGLF4 mediates hyperphosphorylation of cellular elongation factor 1delta (EF-1delta): EF-1delta is universally modified by conserved protein kinases of herpesviruses in mammalian cells.

Translation elongation factor 1delta (EF-1delta) is hyperphosphorylated in various mammalian cells infected with alpha-, beta- and gammaherpesviruses and EF-1delta modification is mediated by viral protein kinases, including UL13 of herpes simplex virus type 1 and UL97 of human cytomegalovirus. In this study, the following is reported. (i) BGLF4 encoded by the prototype gammaherpesvirus Epstein-Barr virus was purified as a fusion protein that was labelled with [gamma-(32)P]ATP and labelling was eliminated by phosphatase. (ii) The ratio of the hyperphosphorylated form of human EF-1delta was increased both in Sf9 cells after infection with baculoviruses expressing GST-BGLF4 fusion proteins and in COS-7 cells after transfection with a BGLF4 expression plasmid. These results indicate that purified BGLF4 possesses protein kinase activity and mediates EF-1delta hyperphosphorylation. These data also support the hypothesis that the protein kinases that are conserved by herpesviruses universally mediate EF-1delta modification in mammalian cells.