Co-deteriorations of anisotropic extracellular matrix arrangement and intrinsic mechanical property in c-src deficient osteopetrotic mouse femur.

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.

Discovery of anti-varicella zoster virus activity of polyether antibiotic CP-44161.

In search for new anti-varicella zoster virus (VZV) compounds with new mechanism of action, we applied a DNA hybridization assay (dot blot method) for screening. Using this method, we screened microbial products and found the polyether compound CP-44161 from the culture broth of an actinomycete strain. CP-44161 was previously reported as an anticoccidal agent, but there has been no claim of its antiviral activities. CP-44161 showed strong anti-VZV activity against pOka strain by plaque reduction assay. Moreover, CP-44161 showed lower cytotoxicity than other antiviral polyethers, such as monensin and nigericin. Its better safety margin and strong anti-VZV properties make it a good candidate for a new anti-VZV agent.

Anti-herpes virus activity of polyether antibiotic CP-44161 in vivo.

In the previous study, we discovered a polyether antibiotic CP-44161, which was reported earlier as an anticoccidal agent, as an anti-varicella zoster virus compound. In this study, we demonstrated that CP-44161 had a very strong and broad anti-herpes virus activities against herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) in vitro. To determine the antiviral activity of CP-44161 in vivo, we examined its effect on the cutaneous HSV-2 infection model in Balb/c mice. CP-44161 showed inhibitory effect on lesion development as well as acyclovir (ACV) when the treatment was started from day 3. Meanwhile, in case the start of treatment was delayed until day 4, when ACV was no longer effective, the effectiveness of CP-44161 still remained. In this model, CP-44161 also showed inhibitory effect on the proliferation of HSV-2 DNA in dorsal root ganglia. This is the first article to report that polyether antibiotics can be effective on viral infection in vivo.

Role of the varicella-zoster virus gene product encoded by open reading frame 35 in viral replication in vitro and in differentiated human skin and T cells in vivo.

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 rOkaDelta35(#1) and rOkaDelta35(#2), and replaced ORF35 at a nonnative site, generating two rOkaDelta35/35@Avr mutants. ORF35 Flag-tagged recombinants were made by inserting ORF35-Flag at the nonnative Avr site as the only copy of ORF35, yielding rOkaDelta35/35Flag@Avr, or as a second copy, yielding rOka35Flag@Avr. Replication of rOkaDelta35 viruses was diminished in melanoma and Vero cells in a 6-day analysis of growth kinetics. Plaque sizes of rOkaDelta35 mutants were significantly smaller than those of rOka in melanoma cells. Infection of melanoma cells with rOkaDelta35 mutants was associated with disrupted cell fusion and polykaryocyte formation. The small plaque phenotype was not corrected by growth of rOkaDelta35 mutants in melanoma cells expressing the major VZV glycoprotein E, gE. The rOkaDelta35/35@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.

FR177391, a new anti-hyperlipidemic agent from Serratia. I. Taxonomy, fermentation, isolation, physico-chemical properties, structure elucidation and biological activities.

In the course of screening for a new anti-hyperlipidemic agent from microbial products, we found that FR177391, produced by Serratia liquefaciens No. 1821, alleviated the decrease in lipid droplet formation in differentiated 3T3-L1 adipocyte cells, induced by the addition of tumor necrosis factor-alpha. Structural elucidation by spectroscopic methods and X-ray crystallographic analysis of its propylamide derivative revealed that FR177391 was a chlorinated macrocyclic lactone.

FR182876, a new microtubule modulator with high water-solubility, from a Streptomyces.

In the course of seeking new anti-tumor drugs, a new microtubule modulator with high water-solubility, FR182876, was isolated from a Streptomyces which also produces FR182877. Even modern spectroscopic methods could not solve the structure of FR182876 due to its structural complexity and chemical instability. Thus, we have combined chemical correlations with spectroscopic methods and determined its structure, which features a highly fused ring system and 3-methylhistidine. The latter is believed to contribute to both solubility in water and activity in promoting tubulin polymerization. FR182876 showed potent cytotoxicity against a panel of cancer cells at concentrations of 28-75 ng/ml.

FR901512, a novel HMG-CoA reductase inhibitor produced by an agonomycetous fungus no. 14919. II. Biological profiles.

FR901512, a new specific inhibitor of HMG-CoA reductase, was isolated from the culture of an agonomycetous fungus No. 14919. FR901512 inhibited cholesterol synthesis from [14C] acetate in Hep G2 cells with an IC50 of 1.0 nM. An increase of cell surface LDL receptors observed on the FR901512 treated human hepatoma cell line Hep G2 cells. Single oral administration of FR901512 strongly inhibited sterol synthesis in rats. Daily oral administration of FR901512 to beagle dogs decreased plasma cholesterol levels.

FR901512, a novel HMG-CoA reductase inhibitor produced by an agonomycetous fungus No. 14919. II. Taxonomy of the producing organism, fermentation, isolation and physico-chemical properties.

Novel compounds FR901512 and FR901516 were isolated from the fermentation broth of agonomycete strain No. 14919. FR901512 and FR901516 possess unique tetralin ring in their structure. These compounds were potent inhibitors of the cholesterol synthesis in human hepatoma cell line Hep G2. FR901512 shows strong 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitory activity with an IC50 value of 0.95 nM.

Requirement of varicella-zoster virus immediate-early 4 protein for viral replication.

Varicella-zoster virus (VZV) is an alphaherpesvirus that causes two diseases, chickenpox and zoster. VZV open 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.

Mutational analysis of open reading frames 62 and 71, encoding the varicella-zoster virus immediate-early transactivating protein, IE62, and effects on replication in vitro and in skin xenografts in the SCID-hu mouse in vivo.

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 Delta 62 (rOka Delta 62) or rOka Delta 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.