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Background: The therapeutic effectiveness of cisplatin, a widely used chemotherapy drug for oral squamous cell carcinoma (OSCC), is often compromised by resistance, making it difficult to predict treatment outcomes. The role of myotubularin and myotubularin-related (MTMR) genes in cisplatin resistance remains unclear. We aimed to elucidate the molecular mechanisms underlying MTMR6 with cisplatin resistance in OSCC., Methods: MTMR6 expression was compared between UMSCC1 and cisplatin-resistant UM-Cis cells. Gain- and loss-of-function experiments involving MTMR6 was performed to evaluate its impact on cisplatin resistance. The regulatory role of hsa-miR-544a on MTMR6 expression was explored via antagomir and miRNA mimic assays. The relationship between MTMR6 protein levels and cisplatin sensitivity was assessed in OSCC patient tissues classified as sensitive or resistant to cisplatin monotherapy. A survival analysis based on The Cancer Genome Atlas (TCGA) head and neck squamous cell carcinoma (HNSCC) dataset was performed to evaluate the correlation between MTMR6 expression and patient outcomes following cisplatin treatment. In vivo cisplatin resistance was examined using mouse xenografts derived from MTMR6-knockdown UMSCC1 cells., Results: MTMR6 expression was markedly reduced in cisplatin-resistant UM-Cis cells compared to UMSCC1 cells. Functional analyses revealed that modulating MTMR6 activity alters cisplatin resistance. A luciferase assay confirmed that hsa-miR-544a regulates MTMR6 gene expression. Additionally, antagomir and miRNA mimics demonstrated that hsa-miR-544a enhances cisplatin resistance by suppressing MTMR6 expression. In OSCC patient tissues, higher MTMR6 protein levels were associated with cisplatin sensitivity, while cisplatin-resistant tissues had lower MTMR6 expression. Survival analysis of the TCGA HNSCC dataset indicated that low MTMR6 expression correlates with poorer outcomes in cisplatin-treated patients compared to those with high MTMR6 expression. Mouse xenografts derived from MTMR6-knockdown UMSCC1 cells exhibited increased resistance to cisplatin compared to controls., Conclusion: Assessing mRNA levels of MTMR6 and has-miR-544a in biopsy samples could help predict cisplatin responsiveness in OSCC., Competing Interests: Declarations. Ethics approval and consent to participate: Human tissue specimens were used after receiving written, informed consent from the patients and approval from the Institutional Research Ethics Committee of Kyungpook National University Hospital (KNUH201704011) with the basic principles of the Declaration of Helsinki. All experimental protocols with mice followed the ARRIVE guidelines (Animal Research: Reporting of In Vivo Experiments) and were approved by the Animal Ethics Committee of Kyungpook National University (2017-94-2). Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Foamy virus (FV) is a retrovirus with a safer integration profile than other retroviruses, rendering it appealing for gene therapy. Prototype FV (PFV) vector systems have been devised to yield high-titer vectors carrying large transgenes. Subsequent iterations of PFV vectors have been engineered to be replication-incompetent, enhancing their safety. A third generation PFV vector system, composed of four plasmids, has been adapted to accommodate large transgenes. Additionally, a novel dual-vector system shows promise for convenient and efficient gene delivery, particularly with the forthcoming development of stable producer cell lines expressing PFV Env. FVs exhibit a broad host spectrum due to the ubiquitous presence of the host factor, heparan sulfate (HS), on their surface. The receptor-binding domain (RBD) of FV Env proteins plays a crucial role in binding to the host cell HS. The FV vector system has been employed in hematopoietic stem cell (HSC) gene therapy to address monogenic diseases in dog and mouse models. In addition, FV vectors safely and efficiently deliver anti-HIV transgenes to HSCs, and vectors carrying HIV epitopes successfully induce antibodies against HIV, offering the promise of anti-HIV gene therapy and vaccine development. In this review, we delve into the development and utilization of FV vector systems, emphasizing their unique advantages in gene therapy, including their non-pathogenic nature, broad host tropism, large transgene capacity, and persistence in resting cells. Furthermore, we discuss the potential of FV vectors in tackling current challenges in gene therapy and their viability as valuable tools for treating genetic diseases., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Foamy viruses (FVs) are generally recognized as non-pathogenic, often causing asymptomatic or mild symptoms in infections. Leveraging these unique characteristics, FV vectors hold significant promise for applications in gene therapy. This study introduces a novel platform technology using a pseudo-virus with single-round infectivity. In contrast to previous vector approaches, we developed a technique employing only two vectors, pcHFV lacking Env and pCMV-Env, to introduce the desired genes into target cells. Our investigation demonstrated the efficacy of the prototype foamy virus (PFV) dual-vector system in producing viruses and delivering transgenes into host cells. To optimize viral production, we incorporated the codon-optimized Env (optEnv) gene in pCMV-Env and the Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE) at the 3' end of the transgene in the transfer vector. Consequently, the use of optEnv led to a significant enhancement in transgene expression in host cells. Additionally, the WPRE exhibited an enhancing effect. Furthermore, the introduced EGFP transgene was present in host cells for a month. In an effort to expand transgene capacity, we further streamlined the viral vector, anticipating the delivery of approximately 4.3 kbp of genes through our PFV dual-vector system. This study underscores the potential of PFVs as an alternative to lentiviruses or other retroviruses in the realm of gene therapy.

Among several anti-cancer therapies, chemotherapy can be used regardless of the stage of the disease. However, development of anti-cancer agents from potential chemicals must be executed very cautiously because of several problems, such as safety, drug resistance, and continuous administration. Most chemotherapeutics selectively cause cancer cells to undergo apoptosis. In this study, we tested the effects of a novel chemical, the benzothiazole derivative N-[2-[(3,5-dimethyl-1,2-oxazol-4-yl)methylsulfanyl]-1,3-benzothiazol-6-yl]-4-oxocyclohexane-1-carboxamide (PB11) on the human cell lines U87 (glioblastoma), and HeLa (cervix cancer). It was observed that this chemical was highly cytotoxic for these cells (IC50s < 50 nM). In addition, even 40 nM PB11 induced the classical apoptotic symptoms of DNA fragmentation and nuclear condensation. The increase of caspase-3 and -9 activities also indicated an increased rate of apoptosis, which was further confirmed via Western blotting analysis of apoptosis-associated proteins. Accordingly, PB11 treatment up-regulated the cellular levels of caspase-3 and cytochrome-c, whereas it down-regulated PI3K and AKT. These results suggest that PB11 induces cytotoxicity and apoptosis in cancer cells by suppressing the PI3K/AKT signaling pathways and, thus, may serve as an anti-cancer therapeutic.

Due to the broad host suitability of viral vectors and their high gene delivery capacity, many researchers are focusing on viral vector-mediated gene therapy. Among the retroviruses, foamy viruses have been considered potential gene therapy vectors because of their non-pathogenicity. To date, the prototype foamy virus is the only retrovirus that has a high-resolution structure of intasomes, nucleoprotein complexes formed by integrase, and viral DNA. The integration of viral DNA into the host chromosome is an essential step for viral vector development. This process is mediated by virally encoded integrase, which catalyzes unique chemical reactions. Additionally, recent studies on foamy virus integrase elucidated the catalytic functions of its three distinct domains and their effect on viral pathogenicity. This review focuses on recent advancements in biochemical, structural, and functional studies of foamy virus integrase for gene therapy vector research.

Interferon-induced transmembrane (IFITM) proteins as host restriction factors are known to inhibit the replication of several viruses. In this study, transient IFITM expression vectors were used to investigate whether IFITMs inhibit feline foamy viral (FFV) replication and which step of viral replication is inhibited. In our studies, viral production was significantly reduced when cells were infected with FFV at almost same times such as -3, 0, or 3 h post-transfection with IFITM vector. However viral production was not reduced even though cells were infected with FFV at 3 or 6 days post-transfection when production of IFITM proteins was maximized. Considering that IFITM expression was maximized at 3 days post-transfection, the stage of viral replication inhibited by IFITM appears to be the late step of viral replication. Moreover, the viral Gag proteins detected in the virus-infected cell lysates were proportionally correlated with viral titer of the culture supernatants. Therefore, it is likely that IFITMs can restrict production of FFV at the late step of viral replication., Competing Interests: No potential conflict of interest was reported by the author(s)., (© 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.)

Foamy viruses (FV) are retroviruses belonging to the Spumaretrovirinae subfamily. They are non-pathogenic viruses endemic in several mammalian hosts like non-human primates, felines, bovines, and equines. Retroviral DNA integration is a mandatory step and constitutes a prime target for antiretroviral therapy. This activity, conserved among retroviruses and long terminal repeat (LTR) retrotransposons, involves a viral nucleoprotein complex called intasome. In the last decade, a plethora of structural insights on retroviral DNA integration arose from the study of FV. Here, we review the biochemistry and the structural features of the FV integration apparatus and will also discuss the mechanism of action of strand transfer inhibitors.

DD(35)E motif in catalytic core domain (CCD) of integrase (IN) is extremely involved in retroviral integration step. Here, nine single residue mutants of feline foamy virus (FFV) IN were generated to study their effects on IN activities and on viral replication. As expected, mutations in the highly conserved D107, D164, and E200 residues abolished all IN catalytic activities (3'-end processing, strand transfer, and disintegration) as well as viral infectivity by blocking viral DNA integration into cellular DNA. However, Q165, Y191, and S195 mutants, which are located closely to DDE motif were observed to have diverse levels of enzymatic activities, compared to those of the wild type IN. Their mutant viruses produced by one-cycle transfection showed different infectivity on their natural host cells. Therefore, it is likely that effects of single residue mutation at DDE motif is critical on viral replication depending on the position of the residues.

Immunosuppressive drugs are used to make the body less likely to reject transplanted organs or to treat autoimmune diseases. In this study, five immunosuppressive drugs including two glucocorticoids (dexamethasone and prednisolone), one calcineurin inhibitor (cyclosporin A), one non-steroid anti-inflammatory drug (aspirin), and one antimetabolite (methotrexate) were tested for their effects on viral proliferation using feline foamy virus (FFV). The five drugs had different cytotoxic effects on the Crandell-Ress feline kidney (CRFK) cells, the natural host cell of FFV. Dexamethasone-pretreated CRFK cells were susceptible to FFV infection, but pretreatment with prednisolone, cyclosporin A, aspirin, and methotrexate showed obvious inhibitory effects on FFV proliferation, by reducing viral production to 29.8-83.8% of that of an untreated control. These results were supported by western blot, which detected viral Gag structural protein in the infected cell lysate. As our results showed a correlation between immunosuppressive drugs and susceptibility to viral infections, it is proposed that immune-compromised individuals who are using immune-suppressive drugs may be especially vulnerable to viral infection originated from pets.

Integrase (IN) is an essential enzyme in retroviral life cycle. It mediates viral cDNA integration into host cellular DNA. Feline foamy virus (FFV) is a member of the Spumavirus subfamily of Retroviridae. Recently, its life cycle has been proposed to be different from other retroviruses. Despite this important finding, FFV IN is not understood clearly. Here, we constructed point mutations in FFV IN C-terminal domain (CTD) to obtain a clear understanding of its integration mechanism. Mutation of the amino acid residues in FFV IN CTD interacting with target DNA reduced both IN enzymatic activities in vitro and viral productions in infected cells. Especially, the mutants, R307 and K340, made viral DNA integration less efficient and allowed accumulation of more unintegrated viral DNA, thereby suppressing viral replication. Therefore, we suggest that the CTD residues interacting with the target DNA play a significant role in viral DNA integration and replication., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Wine fermentations using rice media containing either Monascus koji or rice nuruk were performed and fermentative characteristics based on the koji type were investigated. Cultivations were performed in a 20 °C room in a 20 L bottle with the rice media that included Monascus rice koji at both 20 and 30%, or rice nuruk at 20%. After 22 days of cultivation, the ethanol yield reached 14.2-14.6% (v/v) for M. koji and 16.5% (v/v) for rice nuruk. This lower yield with use of M. koji was thought to be due to rapid cell concentration decreases in the later stage. Total amounts of organic acids and volatile compounds in fermentations using M. koji were 166-172 and 1779-1874 mg/L, respectively, being 8.7-12.9% and 46.3-54.1% higher than with use of rice nuruk. With M. koji, a high quality rice wine was produced with high levels of volatile compounds and monacolin K., Competing Interests: Compliance with ethical standardsThe authors declare no conflict of interest.

The foamy viruses are currently considered essential for development as vectors for gene delivery. Previous studies demonstrated that prototype foamy virus (PFV) can infect and replicate prevalently in a variety of cell types for its exclusive replication strategy. However, the virus-host interaction, especially PFV-transportin3 (TNPO3), is still poorly understood. In our investigation of the role of TNPO3 in PFV infection, we found lower virus production in TNPO3 knockdown (KD) cells compared with wild-type 293T cells. PCR analysis revealed that viral DNAs were mostly altered to circular forms: both 1-long terminal repeat (1-LTR) and 2-LTR in TNPO3 KD cells. We therefore suggest that TNPO3 is required for successful PFV replication, at least at/after the nuclear entry step of viral DNA. These findings highlight the obscure mysteries of PFV-host interaction and the requirement of TNPO3 for productive infection of PFV in 293T cells.

Reverse transcription of viral RNA and the subsequent integration of reverse transcripts are the classical early events of the HIV-1 life-cycle. Simultaneously, abundant unintegrated DNAs (uDNAs), are formed in cells ubiquitously. The uDNAs either undergo recombination or degradation or persist inactively for long periods in the nucleus as future resources. Among them, 2-LTR circles are considered a dead-end for viral spread. Their contribution to the HIV-1 infection is still poorly understood. Nevertheless, the preintegration transcription of the aberrant DNAs and the consequent alterations of cellular factors have already been reported. Since the major fate of the viral genome is to persist as episomal DNA, precise characterization is required for studying the biology of HIV-1. This review compiles the biochemical and genetic updates on uDNA in the HIV-1 life cycle and could provide direction to further study of their roles in HIV-1 replication and application in HIV-1 pathogenesis.

Retroviruses must integrate their cDNA into the host genome to generate proviruses. Viral DNA-protein complexes interact with cellular proteins and produce pre-integration complexes, which carry the viral genome and cross the nuclear pore channel to enter the nucleus and integrate viral DNA into host chromosomal DNA. If the reverse transcripts fail to integrate, linear or circular DNA species such as 1- and 2-long terminal repeats are generated. Such complexes encounter numerous cellular proteins in the cytoplasm, which restrict viral infection and protect the nucleus. To overcome host cell defenses, the pathogens have evolved several evasion strategies. Viral proteins often contain nuclear localization signals, allowing entry into the nucleus. Among more than 1000 proteins identified as required for HIV infection by RNA interference screening, karyopherins, cleavage and polyadenylation specific factor 6, and nucleoporins have been predominantly studied. This review discusses current opinions about the synergistic relationship between the viral and cellular factors involved in nuclear import, with focus on the unveiled mysteries of the host-pathogen interaction, and highlights novel approaches to pinpoint therapeutic targets.

Transportin 3 (TNPO3) is a member of the importin-ß superfamily proteins. Despite numerous studies, the exact molecular mechanism of TNPO3 in retroviral infection is still controversial. Here, we provide evidence for the role and mechanism of TNPO3 in the replication of prototype foamy virus (PFV). Our findings revealed that PFV infection was reduced 2-fold by knockdown (KD) of TNPO3. However, late stage of viral replication including transcription, translation, viral assembly, and release was not influenced. The differential cellular localization of PFV integrase (IN) in KD cells pinpointed a remarkable reduction of viral replication at the nuclear import step. We also found that TNPO3 interacted with PFV IN but not with Gag, suggesting that IN-TNPO3 interaction is important for nuclear import of PFV pre-integration complex. Our report enlightens the mechanism of PFV interaction with TNPO3 and support ongoing research on PFV as a promising safe vector for gene therapy.

We identified four basic amino acid residues as nuclear localization signals (NLS) in the C-terminal domain of the prototype foamy viral (PFV) integrase (IN) protein that were essential for viral replication. We constructed seven point mutants in the C-terminal domain by changing the lysine and arginine at residues 305, 308, 313, 315, 318, 324, and 329 to threonine or proline, respectively, to identify residues conferring NLS activity. Our results showed that mutation of these residues had no effect on expression assembly, release of viral particles, or in vitro recombinant IN enzymatic activity. However, mutations at residues 305 (R → T), 313(R → T), 315(R → P), and 329(R → T) lead to the production of defective viral particles with loss of infectivity, whereas non-defective mutations at residues 308(R → T), 318(K → T), and 324(K → T) did not show any adverse effects on subsequent production or release of viral particles. Sub-cellular fractionation and immunostaining for viral protein PFV-IN and PFV-Gag localization revealed predominant cytoplasmic localization of PFV-IN in defective mutants, whereas cytoplasmic and nuclear localization of PFV-IN was observed in wild type and non-defective mutants. However sub-cellular localization of PFV-Gag resulted in predominant nuclear localization and less presence in the cytoplasm of the wild type and non-defective mutants. But defective mutants showed only nuclear localization of Gag. Therefore, we postulate that four basic arginine residues at 305, 313, 315 and 329 confer the karyoplilic properties of PFV-IN and are essential for successful viral integration and replication.

Successful integration of retroviral DNA into the host chromosome is an essential step for viral replication. The process is mediated by virally encoded integrase (IN) and orchestrated by 3'-end processing and the strand transfer reaction. In vitro reaction conditions, such as substrate specificity, cofactor usage, and cellular binding partners for such reactions by the three distinct domains of prototype foamy viral integrase (PFV-IN) have been described well in several reports. Recent studies on the three-dimensional structure of the interacting complexes between PFV-IN and DNA, cofactors, binding partners, or inhibitors have explored the mechanistic details of such interactions and shown its utilization as an important target to develop anti-retroviral drugs. The presence of a potent, non-transferable nuclear localization signal in the PFV C-terminal domain extends its use as a model for investigating cellular trafficking of large molecular complexes through the nuclear pore complex and also to identify novel cellular targets for such trafficking. This review focuses on recent advancements in the structural analysis and in vitro functional aspects of PFV-IN.

A new manufacturing method for producing a puffed ginseng-rice snack (PGRS) was developed using ginseng powder and map rice flour through a steam and compression process (SCP). The physical and sensory properties of the PGRS were characterized. The pellets for puffing were prepared from ginseng powder and map rice flour. The pellets were subjected to 16, 18, and 20% moisture contents and were puffed at 225, 235, and 245° C. The specific volumes of the PGRSs increased with heating temperature and moisture content. However, the breaking strength of the PGRSs decreased. In addition, the SCP imposed special features in the PGRSs that made them more acceptable. The Hunter L-value increased with heating temperature and moisture content. These results indicate that a PGRS with functional additives could be effectively developed into a functional food with the use of a puffing machine, and that the PGRS shows potential as a new snack product.

We constructed deletion mutants and seven point mutants by polymerase chain reaction to investigate the specificity of feline foamy virus integrase functional domains. Complementation reactions were performed for three enzymatic activities such as 3'-end processing, strand transfer, and disintegration. The complementation reactions with deletion mutants showed several activities for 3'-end processing and strand transfer. The conserved central domain and the combination of the N-terminal or C-terminal domains increased disintegration activity significantly. In the complementation reactions between deletion and point mutants, the combination between D107V and deletion mutants revealed 3'-end processing activities, but the combination with others did not have any activity, including strand transfer activities. Disintegration activity increased evenly, except the combination with glutamic acid 200. These results suggest that an intact central domain mediates enzymatic activities but fails to show these activities in the absence of the N-terminal or C-terminal domains.

An electrically tunable photonic crystal is developed utilizing crystalline colloidal arrays of high refractive index particles. Through modulation of the refractive index of the particle, and the applied electric field, both the bandwidth and position of the photonic bandgap could be tuned. Full color modulation with high optical quality is achieved, which paves a way to develop a novel reflective display., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

We have reported previously that the prototype foamy virus (PFV) integrase (IN) has a strong nuclear localization signal (NLS) in its C-terminal domain, in particular in a region of aa 306-334 including highly karyophilic arginines or lysines at positions 308, 313, 318, 324, and 329. In this study, we used various mutants of the C-terminal domain to further analyze its karyophilic determinants. Plasmids expressing these mutants fused to maltose binding protein (MBP) and enhanced green fluorescent protein (EGFP) were transfected to COS-1 cells and subcellular localization of these fluorescent fusion proteins was determined by fluorescent microscopy. The results revealed that a maximum karyophilicity was exhibited by a region longer than the previously described one of 29 aa (aa 306-334), in particular by a 64 aa region (aa 289-352) with Arg341 and Lys349 as critical determinants.

In order to study biochemical properties, the integrase (IN) protein of feline foamy virus (FFV) was over-expressed from Escherichia coli, purified by two-step chromatography; Talon column and heparin column, and characterized in biochemical aspects. For the three enzymatic reactions of the 3' -processing, strand transfer, and disintegration activities, Mn2+ ion was essentially required as a cofactor. Interestingly, Co2+ and Zn2+ ions were found to act as effective cofactors, while other transition elements such as Ni2+, Cu2+, La3+, Y3+, Cd2+, Li1+, Ba2+, Sr2+, V3+, and so on were not. Regarding to the substrate specificity, FFV IN has low substrate specificities as it cleaved in a significant level prototype foamy virus (PFV) U5 LTR substrate as well as FFV U5 LTR substrate, while PFV IN did not. Finally, the 3' -processing activity was observed in the high concentrations of several solvents such as CHAPS, Glycerol, Tween 20 and Triton X-100, which are generally used for dissolution of chemicals in inhibitor-screening. Therefore, as it is the first report showing biochemical properties, FFV IN is proposed to have low specificities on the use of cofactor and substrate for enzymatic reaction when it is compared with other retroviral INs.

Some enniatins (ENs) reportedly exhibit antiretroviral activities in vivo. The potential inhibitory activities of cyclic hexadepsipeptides such as beauvericin (BEA) and ENs H, I and MK1688 were investigated in vitro against human immunodeficiency virus type-1 (HIV-1) integrase and Moloney murine leukemia virus reverse transcriptase. BEA, EN I and EN MK1688 exhibited strong inhibitory activities against HIV-1 integrase, whereas EN H showed relatively weak activity. None of the examined compounds showed anti-reverse transcriptase activity. BEA was the most effective inhibitor of the tested cyclic hexadepsipeptides in inhibiting HIV-1 integrase. These results indicate the potential of cyclic hexadepsipeptides as a new class of potent inhibitors of HIV-1 integrase.

Enniatins are cyclic peptides isolated from bacteria, fungi, and plants, with numerous biological effects on animal systems. Recently, we have reported that certain enniatins (ENs), such as EN H and EN MK1688, have cytotoxic effects on several adenocarcinoma cell lines. In an effort to understand the mechanism behind their cytotoxicity, we investigated whether ENs can induce apoptosis in human colorectal carcinoma cells (HCT-15 cells). Treatment with the ENs H and MK1688 resulted in an alteration of cellular and nuclear morphology, leading to an increase in the number of the cells with apoptotic nuclei (seen as condensed or fragmented nuclei). Furthermore, it was observed that cellular DNA fragmentation increased in a dose-dependent manner in EN treated cells. These cells have elevated activity levels for caspase-3, the enzyme responsible for initiating cell death, compared with the untreated cells. Normal caspase-3 activity levels were observed when Z-VAD-FMK, a caspase inhibitor, was added simultaneously with the ENs. Based on our results, we propose that the new ENs H and MK1688 induce cytotoxicity via an apoptotic pathway.

To analyse the potential karyophilic activity of prototype foamy viruses (PFVs), we expressed the PFV integrase (IN) and its mutants as fusion proteins with enhanced green fluorescence protein. The subcellular localization of the fusion proteins was investigated by fluorescence microscopy. The PFV IN was found to be karyophilic and targeted the fusion protein to the nucleus. Mutational analyses demonstrated that the PFV IN contains a potent but non-transferable nuclear localization signal (NLS) in its C-terminal domain and contains five arginine and lysine residues between amino acids 308 and 329 that are critical for its NLS function.

In order to study functional nucleotides in prototype foamy virus (PFV) DNA on specific recognition by PFV integrase (IN), we designed chimeric U5 long terminal repeat (LTR) DNA substrates by exchanging comparative sequences between human immunodeficiency virus type-1 (HIV-1) and PFV U5 LTRs, and investigated the 3'-end processing reactivity using HIV-1 and PFV INs, respectively. HIV-1 IN recognized the nucleotides present in the fifth and sixth positions at the 3'-end of the substrates more specifically than any other nucleotides in the viral DNA. However, PFV IN recognized the eighth and ninth nucleotides as distinctively as the fifth and sixth nucleotides in the reactions. In addition, none of the nucleotides present in the twelfth, sixteenth, seventeenth, eighteenth, nineteenth, and twentieth positions were not differentially recognized by HIV-1 and PFV INs, respectively. Therefore, our results suggest that the functional nucleotides that are specifically recognized by its own IN in the PFV U5 LTR are different from those in the HIV-1 U5 LTR in aspects of the positions and nucleotide sequences. Furthermore, it is proposed that the functional nucleotides related to the specific recognition by retroviral INs are present inside ten nucleotides from the 3'-end of the U5 LTR.

Enniatins (ENs) H, I, and MK1688 and beauvericin (BEA) were purified from concentrated chloroform extracts of Fusarium oxysporum KFCC 11363P submerged cultures using HPLC, and their in vitro cytotoxicities were evaluated against four human carcinoma cell lines (lung, A549; ovarian, SK-OV-3; skin melanoma, SK-MEL-2; and colon, HCT15) using the sulforhodamine B (SRB) method. ENs I and MK1688 inhibited the growth of cancer cell lines most strongly and had similar cytotoxic effects on the tested human cancer cell cultures. The cytotoxicity of ENs I and MK1688 was three- to fourfold higher than that of BEA and EN H. When cultivated in Fusarium-defined medium (FDM), the concentrations of ENs and BEA produced in F. oxysporum KFCC 11363P decreased in the following order: EN MK1688 (0.81 g/L) >EN I (0.55 g/L) >BEA (0.17 g/L) > EN H (0.16 g/L). This study has shown that ENs H, I, and MK1688 exhibit cytotoxicity against certain adenocarcinoma cell lines. The results indicate the need for more investigations into the significance of the biological properties of these new ENs.

HIV-1 integrase catalyzes terminal cleavage at the 3' end of the proviral DNA, removing a pair of bases and causing strand transfer by joining the 3' end to 5'-phosphates in the target DNA. Several aryl 1,3-diketo acids that can inhibit the strand transfer reaction of HIV-1 IN have been identified. Here we synthesized a new series of compounds with a chromone or chromanone ring as conformationally constrained scaffolds of 1,3-diketo acids, and then tested their ability to inhibit HIV-1 IN-mediated strand transfer. All compounds moderately inhibited HIV-1 IN activity, indicating that the conformational restriction of one keto group into a chromone or chromanone ring decreases inhibition of the HIV-1 IN strand transfer.

The production of the entomopathogenic and phytotoxic cyclic depsipeptide beauvericin (BEA) was studied in submerged cultures of Fusarium oxysporum KFCC 11363P isolated in Korea. The influences of various factors on mycelia growth and BEA production were examined in both complete and chemically defined culture media. The mycelia growth and BEA production were highest in Fusarium defined medium. The optimal carbon and nitrogen sources for maximizing BEA production were glucose and NaNO3, respectively. The carbon/ nitrogen ratio for maximal production of BEA was investigated using response surface methodology (RSM). Equations derived by differentiation of the RSM model revealed that the production of BEA was maximal when using 108 mM glucose and 25 mM NaNO3.

Human immunodeficiency virus (HIV) integrase (IN) catalyzes the integration of HIV DNA copy into the host cell DNA. L-Chicoric acid (1) has been found to be one of the most potent HIV-1 integrase inhibitor. Caffeoylglycolic and caffeoylamino acid derivatives' halfmeric structures of L-chicoric acid 2 were synthesized for the purpose of simplifying the structure of L-chicoric acid. Among synthesized, compounds 2c and 3f showed HIV-1 IN inhibitory activities with IC(50) values of 10.5 and 12.0 microM, respectively, comparable to that of parent compound L-chicoric acid (IC(50)=15.7 microM).

The bioassay-directed isolation of a marine brown alga, Ishige okamurae, afforded a carmalol derivative, diphlorethohydroxycarmalol. This compound exhibited inhibitory effects on HIV-1 reverse transcriptase and integrase with IC(50) values of 9.1 microM and 25.2 microM, respectively. However, diphlorethohydroxycarmalol did not show an inhibitory activity against HIV-1 protease. Moreover, diphlorethohydroxycarmalol nonaacetate obtained by acetylation and fucosterol failed to show any inhibitory activity against these viral enzymes.

Ethephon at 50 microM enhanced both root growth and ginsenoside accumulation in ginseng (Panax ginseng C.A. Meyer) adventitious root cultures, but at 100 microM it inhibited only ginsenoside accumulation. Ginsenoside productivity with 50 microM ethephon was the highest at 1.7 mg l(-1) d(-1) after 8 days of elicitation. However, elicitation with 50 microM ethephon and 100 microM methyl jasmonate (MJ) improved productivity (6.3 mg l(-1) d(-1)) whereas elicitation with 100 microM MJ alone gave only 2.9 mg l(-1) d(-1).

Bioassay-directed chromatographic fractionation of an ethyl acetate extract of Gardenia jasminoides (Gardeniae Fructus) afforded a new vanillic acid 4-O-beta-d-(6'-sinapoyl)glucopyranoside (1) and five new quinic acid derivatives, methyl 5-O-caffeoyl-3-O-sinapoylquinate (2), ethyl 5-O-caffeoyl-3-O-sinapoylquinate (3), methyl 5-O-caffeoyl-4-O-sinapoylquinate (4), ethyl 5-O-caffeoyl-4-O-sinapoylquinate (5), and methyl 3,5-di-O-caffeoyl-4-O-(3-hydroxy-3-methyl)glutaroylquinate (6), together with three known quinic acid derivatives, two flavonoids, two iridoids, and two phenolic compounds. The structures of new compounds were elucidated by the aid of spectroscopic methods. These compounds were assessed for antioxidant activity using three different cell-free bioassay systems and for HIV-1 integrase inhibitory activity. Five new quinic acid derivatives showed potent DPPH radical scavenging, superoxide anion scavenging, and lipid peroxidation inhibition activities. These new quinic acid derivatives also exhibited HIV-1 integrase inhibitory activity.

Retroviral integrases insert viral DNA into target DNA. In this process they recognize their own DNA specifically via functional domains. In order to analyze these functional domains, we constructed six chimeric integrases by swapping domains between HIV-1 and HFV integrases, and two point mutants of HFV integrase. Chimeric integrases with the central domain of HIV-1 integrase had strand transfer and disintegration activities, in agreement with the idea that the central domain determines viral DNA specificity and has catalytic activity. On the other hand, chimeric integrases with the central domain of HFV integrase did not have any enzymatic activity apart from FFH that had weak disintegration activity, suggesting that the central domain of HFV integrase was defective catalytically or structurally. However, these inactive chimeras were efficiently complemented by the point mutants (D164A and E200A) of HFV integrase, indicating that the central domain of HFV integrase possesses potential enzymatic activity but is not able to recognize viral or target DNA without the help of its homologous N-terminal and C-terminal domains.

Squalene synthase (SS) catalyzes the first committed step in sterol and triterpenoid biosynthesis. Transgenic Eleutherococcus senticosus Rupr. and Maxim. plants were generated by introducing an SS-encoding gene derived from Panax ginseng (PgSS1) together with genes expressing hygromycin phosphotransferase and green fluorescent protein (GFP) through Agrobacterium-mediated transformation. Early globular embryo clusters developing from the embryogenic callus were used for Agrobacterium-mediated transformation. Transformants were selected on Murashige Skoog medium containing 25 mg/L hygromycin. Hygromycin-resistant somatic embryos developed into plants after the cotyledonary embryos were treated with 14.4 microM gibberellic acid. Transformation was confirmed by polymerase chain reaction, Southern, and GFP analyses. The SS enzyme activity of the transgenic plants was up to 3-fold higher than that of wild-type plants. In addition, GC-MS and HPLC analysis revealed that phytosterols (beta-sitosterol and stigmasterol) as well as triterpene saponins (ciwujianosides B (1), C(1) (2), C(2) (3), C(3) (4), C(4) (5), D(1) (6) and D(2) (7)) levels in transgenic E. senticosus were increased by 2- to 2.5-fold. These results suggest that the metabolic engineering of E. senticosus to enhance production of phytosterols and triterpenoids by introducing the PgSS1 gene was successfully achieved by Agrobacterium-mediated genetic transformation.

Roots of Panax ginseng, one of the most famous medicinal plants, contain various phytosterols and bioactive triterpene saponins (ginsenosides). In P. ginseng, phytosterols and triterpenes share the common biosynthetic intermediate, squalene. Here, we investigate the regulatory role of Panax ginseng squalene synthase (PgSS1) on the biosynthesis of phytosterols and triterpene saponins. PgSS1 transcripts are expressed ubiquitously in the various plant tissues, but higher in shoot apex and root. The transcript levels of PgSS1 increased markedly in the adventitious roots during 12- to 96-h period after metyl jasmonate (MeJA) treatment; MeJA treatment induced the activation of the transcripts of squalene epoxidase (SE), beta-amyrin synthase (bAS), but not cycloartenol synthase (CAS). Unlike MeJA treatment, overexpression of PgSS1 in adventitious roots of transgenic P. ginseng was followed by the up-regulation of all the downstream genes tested, such as SE, bAS, and CAS. The enhanced activity of PgSS1 enzyme resulted in remarkable increase of phytosterols as well as ginsenoside contents. These results demonstrate that PgSS1 is a key regulatory enzyme not only for phytosterol but also for triterpene biosynthesis and overexpressing of PgSS1 confers the hyperproduction of triterpene saponins to P. ginseng.

DA-125, a novel derivative of adriamycin, is known for its anti-cancer activity. In this study, the inhibitory mechanism of DA-125 on topoisomerase was investigated in the simian virus 40 (SV40) replicating CV-1 cell by studying the SV40 DNA replication intermediates and DNA-topoisomerase complexes. DNA-protein complexes that were formed in the drug-treated cells were quantitated by using a glass filter assay. SV40 DNA replication intermediates that were accumulated in the drug-treated CV-1 cell were analyzed in a high resolution gel. DA-125 did not accumulate B-dimers of SV40 DNA replication intermediates which were found in the adriamycin-treated CV-1 cells. DA-125 induced a dose-dependent formation of the DNA-protein complexes, while adriamycin did not. When adriamycin and etoposide (VP16) were added to the SV40-infected cells at the same time, adriamycin blocked the formation of the DNA-protein complexes induced by VP16 in a dose-dependent manner. However, DA-125 blocked the formation of the DNA-protein complexes induced by VP16 up to the maximum level of the DNA-protein complexes that were induced by DA-125 alone. Adriamycin and DA-125 did not inhibit the formation of the DNA-protein complexes that were caused by camptothecin, a known topoisomerase I poison. DA-125 is bifunctional in inhibiting topoisomerase II because it simultaneously has the properties of the topoisomerase II poison and the DNA intercalator. As a topoisomerase II poison, DA-125 alone induced dose-dependent formation of the DNA-protein complexes. However, as a DNA intercalator, it quantitatively inhibited the formation of the DNA-protein complexes induced by a strong topoisomerase II poison VP16. Furthermore considering that the levels of the DNA-protein complex induced by VP16 were decreased by DA-125 in terms of the topoisomerase II poison, we suggest that DA-125 has a higher affinity to the drug-binding sites of DNA than VP16 has.

HIV-1 integrase (IN) is an essential enzyme for retroviral replication and a rational target for the design of anti-AIDS drugs. In the present study, we have designed, synthesized and tested a series of caffeoyl naphthalenesulfonamide derivatives as HIV integrase inhibitors. Among these compounds, we found that HIV integrase inhibitory activities of compounds III-3 and III-4 were more potent than L-chicoric acid (IC(50)=11.8 microg/mL) and others were comparable to L-chicoric acid. Furthermore, the structure-activity relationships of these compounds were studied. The information gathered from this paper will be useful in the development and design of HIV-1 integrase inhibitors in the future.

Retroviral integrase plays an important role in choosing host chromosomal sites for integration of the cDNA copy of the viral genome. The domain responsible for target site selection has been previously mapped to the central core of the protein (amino acid residues 49-238). Chimeric integrases between human immunodeficiency virus type 1 (HIV-1) and feline immunodeficiency virus (FIV) were prepared to examine the involvement of a nonspecific DNA-binding region (residues 213-266) and certain alpha helices within the core domain in target site selection. Determination of the distribution and frequency of integration events of the chimeric integrases narrowed the target site-specifying motif to within residues 49-187 and showed that alpha 3 and alpha 4 helices (residues 123-166) were not involved in target site selection. Furthermore, the chimera with the alpha 2 helix (residues 118-121) of FIV identity displayed characteristic integration events from both HIV-1 and FIV integrases. The results indicate that the alpha 2 helix plays a role in target site preference as either part of a larger or multiple target site-specifying motif.

Human immunodeficiency virus type 1 (HIV-1) integrase is an essential enzyme in the life cycle of the virus. It is responsible for catalyzing the insertion of the viral genome into the host cell chromosome. This integrase is an attractive target for the design of a HIV antiviral drug, because integrase has no human counterpart. In order to know the interaction mode of HIV-1 integrase with its inhibitor, we investigated the effect of the inhibitor, baicalein, on the conformation of the HIV-1 integrase catalytic domain [IN-(50-212/F185K)] using fluorescence and circular dichroism (CD) spectroscopy. We found that baicalein binds to the hydrophobic region of the HIV-1 integrase catalytic core domain. This binding of baicalein induces the conformational change of the enzyme. We also found that the binding ratio of baicalein to the HIV-1 integrase catalytic domain is 2:1.

Seven phenylpropanoid glycosides named acteoside (1), acteoside isomer (2), leucosceptoside A (3), plantainoside C (4), jionoside D (5), martynoside (6), and isomartynoside (7) were isolated from Clerodendron trichotomum. Compounds 1 and 2 showed potent inhibitory activities against HIV-1 integrase with IC50 values of 7.8 +/- 3.6 and 13.7 +/- 6.0 microM, respectively.

The phytochemical study of the aerial parts of Aster scaber Thunb. (Asteraceae) yielded a new caffeoyl quinic acid, (-) 3,5-dicaffeoyl-muco-quinic acid (2) and three known compounds, (-) 3,5-dicaffeoyl quinic acid (1), (-) 4,5-dicaffeoyl quinic acid (3), (-) 5-caffeoyl quinic acid (4). The structures were established by high resolution spectroscopic methods. The antiviral effects against HIV-1 integrase of the compounds was evaluated. (-) 3,5-Dicaffeoyl-muco-quinic acid (2) exhibited potent antiviral activity with an IC50 value of 7.0 +/- 1.3 microg/ml.

Caffeoylglucosides, which have a glucose ring as a central linker, were synthesized from methyl D-glucosides, and their anti-HIV-1 activities were tested. Among them, four dicaffeoylglucosides (IC50 = 29.1+/-35.1 microM), 6a, 6b, 9b and 10b, showed HIV-1 integrase inhibitory activity as potent as L-chicoric acid.

The human immunodeficiency virus type-1 (HIV-1) integrase (IN) mediates insertion of viral DNA into human DNA, which is an essential step in the viral life cycle. In order to study minimal core domain in HIV-1 IN protein, we constructed nine deletion mutants by using PCR amplification. The constructs were expressed in Escherichia coli, and the proteins were subsequently purified and analyzed in terms of biological activity such as enzymatic and DNA-binding activities. The mutant INs with an N-terminal or C-terminal deletion showed strong disintegration activity though they failed to show endonucleolytic and strand transfer activities, indicating that the disintegration reaction does not require the fine structure of the HIV-1 IN protein. In the DNA-binding analysis using gel mobility shift assay and UV cross-linking method, it was found that both the central and C-terminal domains are essential for proper DNA-IN protein interaction although the central or C-terminal domain alone was able to be in close contact with DNA substrate. Therefore, our results suggest that the C-terminal domain act as a DNA-holding motive, which leads to proper interaction for enzymatic reaction between the IN protein and DNA.

We have been screening anti-HIV integrase compounds from Korean medicinal plants by using an in vitro assay system which is mainly composed of recombinant human immunodeficiency virus type 1 integrase and radiolabeled oligonucleotides. From the above screening, the aqueous methanolic extract of the roots of Agastache rugosa exhibited a significant activity. Bioactivity-guided chromatographic fractionation of the methanolic extract resulted in the isolation of rosmarinic acid. The structure of the compound was determined by spectroscopic data and by the comparison with the reported values. The IC50 of the rosmarinic acid was approximately 10 microg/ml against HIV integrase.

The integration activity of human immunodeficiency virus type-1 (HIV-1) integrase was characterized in vitro by using pre-processed oligonucleotide substrates. The highest level of integration activity was found at pH 6.5 to 7.0, while the endonucleolytic activity was highest at pH 7.4 to 8.0. Although the endonucleolytic and integration reactions are consecutive in retroviral integration, our result indicates that the optimal conditions of the two reactions are quite different. In addition, it is suggested that the endonucleolytic and integration steps can be separated by control of the cellular physiological state in retroviral therapy. Strong integration was detected in the presence of 0.5-10 mM Mn2+ ion, but weak integration at around 10 mM Mg2+ ion. This observation explains that the Mn2+ ion is preferred to the Mg2+ ion as a cofactor in the integration reaction. Although there was no sequence-specificity in the integration site of the target DNA, integration was found to frequently occur at particular regions of the target DNA. Furthermore, the mutant integrases such as Asp116, Ser147, and Glu152, which had been reported previously, were shown to lose integration activity completely, indicating that these residues are critically involved in catalytic action.

The human immunodeficiency virus type 1 (HIV-1) and human foamy virus (HFV) integrase proteins were overexpressed in Escherichia coli, and purified to a near homogeneity by one- or two-step purification scheme. The endonucleolytic, integration, and disintegration activities for the HIV-1 and HFV integrases were characterized in vitro. The endonucleolytic activities for the HIV-1 and HFV integrases were found only on their own substrates, respectively, indicating that the cognate U5 LTR sequences in the substrates is critical for specific cleavage. However, the integration and disintegration activities showed less specificity on the substrate usage. Our results suggest that the disintegration activity have more preference for substrates based on Y-shaped structure rather than on viral donor DNA sequence.

Bioassay-directed chromatographic fractionation of an ethyl acetate extract of the leaves of Acer okamotoanum using HIV-1 integrase afforded a new acylated flavonol glycoside, quercetin 3-O-(2",6"-O-digalloyl)-beta-D-galactopyranoside (1), together with six known flavonol glycosides and three known phenolic compounds. The structure of the new compound was determined by spectroscopic methods. The most active compounds were quercetin 3-O-(2"-galloyl)-alpha-L-arabinopyranoside (6) and 1, which exhibited IC50 values of 18.1 +/- 1.3 and 24.2 +/- 6.6 micrograms/mL, respectively, against HIV-1 integrase.