Newantitumor substances, FR901463, FR901464 and FR901465 were isolated from the culture broth of a bacterium of Pseudomonas sp. No.2663. FR901463, FR901464 and FR901465 remarkably enhanced the transcriptional activity of the promoter of SV40 DNAvirus. Further, these compounds exhibited potent antitumor activities against murine and human tumor cell lines in vitro.

The varicella-zoster virus (VZV) genome has unique long (U L ) and unique short (U S ) segments which are flanked by internal repeat (IR) and terminal repeat (TR) sequences. The immediate-early 62 (IE62) protein, encoded by open reading frame 62 (ORF62) and ORF71 in these repeats, is the major VZV transactivating protein. Mutational analyses were done with VZV cosmids generated from parent Oka (pOka), a low-passage clinical isolate, and repair experiments were done with ORF62 from pOka and vaccine Oka (vOka), which is derived from pOka. Transfections using VZV cosmids from which ORF62, ORF71, or the ORF62/71 gene pair was deleted showed that VZV replication required at least one copy of ORF62. The insertion of ORF62 from pOka or vOka into a nonnative site in U S allowed VZV replication in cell culture in vitro, although the plaque size and yields of infectious virus were decreased. Targeted mutations in binding sites reported to affect interaction with IE4 protein and a putative ORF9 protein binding site were not lethal. Single deletions of ORF62 or ORF71 from cosmids permitted recovery of infectious virus, but recombination events repaired the defective repeat region in some progeny viruses, as verified by PCR and Southern hybridization. VZV infectivity in skin xenografts in the SCID-hu model required ORF62 expression; mixtures of single-copy recombinant Oka⌬62 (rOka⌬62) or rOka⌬71 and repaired rOka generated by recombination of the single-copy deletion mutants were detected in some skin implants. Although insertion of ORF62 into the nonnative site permitted replication in cell culture, ORF62 expression from its native site was necessary for cell-cell spread in differentiated human skin tissues in vivo.Varicella-zoster virus (VZV) belongs to the alphaherpesvirus subfamily of the Herpesviridae. VZV is the causative agent of varicella, which is characterized by cell-associated viremia and a cutaneous vesicular rash (4). VZV establishes latency in cells within sensory ganglia during primary infection. VZV reactivation from latency results in herpes zoster, a localized skin rash in the distribution of nerves from the affected ganglion. VZV is the first human herpesvirus for which a vaccine has been developed to prevent primary infection (58). This live attenuated varicella vaccine was created by serial passage of a wild-type clinical isolate, the parent Oka (pOka) strain, in guinea pig embryo cells and human fibroblasts to generate the varicella vaccine virus (vOka).The VZV genome consists of approximately 125 kb and has at least 70 unique open reading frames (ORFs) (52). As is characteristic of herpesviruses, the double-stranded DNA genome has unique long (U L ) and unique short (U S ) segments which are flanked by internal repeat (IR) and terminal repeat (TR) sequences. Three duplicated genes, ORF62/71, ORF63/ 70, and ORF64/69, are located in repeats at each end of the U S segment. The two VZV origins of replication, designated OriS, are also located in these repeat regions. The immediate-early 62 (IE62) protein,...

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Using the characteristic morphological changes of mammaliancells, we screened novel antimitotic substances and found that a strain of Streptomyces sp. No.9885 produced FR182877. This substance was isolated from the culture broth by ethyl acetate extraction, silica gel column chromatography and ODScolumn chromatography Structural studies on FR1 82877 suggested that it had a unique hexacyclic structure encompassing its highly strained double bond. FR182877 exhibited potent antitumor activities against murine ascitic tumor and solid tumor in vivo.

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Osteopetrotic bone shows dissociation between bone mineral density (BMD) and bone strength. In this study, volumetric BMD; preferential orientation of the extracellular matrix (ECM), which is composed of collagen fibers and apatite crystals as bone material quality; and mechanical properties of the src osteopetrotic and normal mouse femoral cortical bone were analyzed and compared with each other at a bone tissue level. The degree of preferential orientation of ECM along the femoral long axis was significantly decreased in the src mice femur, suggesting deteriorated bone quality. Young's modulus, as a tissue-level mechanical property analyzed by nano-indentation technique along the long bone direction, also was decreased in the src mice cortical femur, in spite of the similar volumetric cortical BMD. To the best of our knowledge, this is the first report to demonstrate the synchronous deterioration of Young's modulus and anisotropic ECM organization in the src osteopetrotic mouse bone. These results indicate that the deterioration of the preferential ECM orientation is one major cause of the impaired mechanical property in the src mouse bone.

Varicella-zoster virus (VZV) is an alphaherpesvirus that causes two diseases, chickenpox and zoster. VZVopen reading frame 4 (ORF4) encodes the immediate-early 4 (IE4) protein, which is conserved among alphaherpesvirus and has transactivation activity in transient transfections. To determine whether the ORF4 gene product is essential for viral replication, we used VZV cosmids to remove ORF4 from the VZV genome. Deleting ORF4 was incompatible with recovery of infectious virus, whereas transfections done by using repaired cosmids with ORF4 inserted at a nonnative site yielded virus. To analyze the functional domain of IE4, we introduced a mutation altering the C-terminal amino acids, KYFKC (K443S), which was designed to disrupt the dimerization of IE4 protein. Transfections with these mutant cosmids yielded no virus, indicating that this KYFKC motif was essential for IE4 function.Varicella zoster virus (VZV) is a ubiquitous human alphaherpesvirus which causes varicella (chickenpox) during primary infection and herpes zoster (shingles) after its reactivation from sites of latency in sensory ganglia (1, 3). Both varicella and zoster continue to be serious public health problems (1, 11). The expression of VZV genes is regulated in a temporal fashion known as the lytic cascade. The product of open reading frame 4 (ORF4) has been shown to have immediate-early regulatory functions, like immediate-early 62 (IE62) and IE63 (4). The IE4 tegument protein is a 51-kDa phosphoprotein that transactivates genes of all three kinetic classes in transient expression systems and enhances IE62 transactivation (4). In turn, IE62, along with the cellular transcription factor USF, activates the ORF4 promoter (7). IE4 protein resembles its herpes simplex virus (HSV) homolog, ICP27, in its C-terminal residues (38% in amino acids 235 to 449) and complements ICP27 functions in this region, but it does not repair full deletions of the ICP27 gene (8, 9). The equine herpesvirus homolog is UL3 (12). Baudoux et al. have recently mapped IE4 domains in transient expression systems (2), demonstrating that the IE4 transactivating function depends on dimerization mediated by a KYFKC peptide in the C-terminal cysteine-rich region, amino acids 443 to 447. Other mutations in an arginine-rich region of the amino terminus, designated Rb, also reduced transactivation independently of dimerization and may be required for IE4 interactions with cellular proteins, including TATA-binding protein, transcription factor IIB, and the p50 and p65 subunits of NF-B (5). IE4 also has KH-like motifs that are involved in RNA binding which are found in conserved forms in alphaherpesvirus homologs and are required for HSV replication (13).The aim of this report was to examine the role of IE4 protein in viral replication by deleting or mutating ORF4 in the context of the viral genome. We showed that ORF4 is an essential gene and that the sequence encoding the C-terminal KYFKC motif in IE4 protein, which mediates dimerization, is required for VZV replication. Effect of ORF4 ...

Although genes related to varicella-zoster virus (VZV) open reading frame 35 (ORF35) are conserved in the herpesviruses, information about their contributions to viral replication and pathogenesis is limited. Using a VZV cosmid system, we deleted ORF35 to produce two null mutants, designated rOka⌬35(#1) and rOka⌬35(#2), and replaced ORF35 at a nonnative site, generating two rOka⌬35/35@Avr mutants. ORF35 Flag-tagged recombinants were made by inserting ORF35-Flag at the nonnative Avr site as the only copy of ORF35, yielding rOka⌬35/35Flag@Avr, or as a second copy, yielding rOka35Flag@Avr. Replication of rOka⌬35 viruses was diminished in melanoma and Vero cells in a 6-day analysis of growth kinetics. Plaque sizes of rOka⌬35 mutants were significantly smaller than those of rOka in melanoma cells. Infection of melanoma cells with rOka⌬35 mutants was associated with disrupted cell fusion and polykaryocyte formation. The small plaque phenotype was not corrected by growth of rOka⌬35 mutants in melanoma cells expressing the major VZV glycoprotein E, gE. The rOka⌬35/35@Avr viruses displayed growth kinetics and plaque morphologies that were indistinguishable from those of rOka. Analysis with ORF35-Flag recombinants showed that the ORF35 gene product localized predominantly to the nuclei of infected cells. Evaluations in the SCIDhu mouse model demonstrated that ORF35 was required for efficient VZV infection of skin and T-cell xenografts, although the decrease in infectivity was most significant in skin. These mutagenesis experiments indicated that ORF35 was dispensable for VZV replication, but deleting ORF35 diminished growth in cultured cells and was associated with attenuated VZV infection of differentiated human skin and T cells in vivo.Varicella-zoster virus (VZV) is an ubiquitous alphaherpesvirus that causes varicella (chicken pox) and herpes zoster (shingles) in the human host (2, 10). The VZV genome is a double-stranded DNA molecule that consists of the unique long (UL) and unique short segments, each of which is flanked by internal repeat and terminal repeat sequences. Complete sequencing of several VZV strains has demonstrated conservation of the linear organization of open reading frames (ORFs) and limited variation in the DNA sequence among isolates from different geographical regions (1,13,16,17,24). Although the VZV genome contains ORFs that are known or predicted to encode more than 70 distinct proteins, functions have been assigned to only about half of these VZV gene products (10). In many cases, the contributions of VZV genes to viral replication are presumed because of their partial sequence homologies with ORFs in herpes simplex virus type 1 (HSV-1), which is the prototype of the alphaherpesviruses (35). While such assumptions have proven to be useful, instances of function or requirements not corresponding to those inferred have also been documented, and HSV and VZV proteins with high apparent homology may not have complementing functions, as illustrated by DNA replication proteins (3, 7, 9, 10).ORF35 is...