Hypericin blocks the function of HSV-1 alkaline nuclease and suppresses viral replication.

ETHNOPHARMACOLOGICAL RELEVANCE: Hypericum perforatum L. has a long history in many countries of being used as a herbal medicine. It is also widely used in Chinese herbal medicine for the treatment of infections. Hypericin, a main component extracted from Hypericum perforatum L., has attracted the attention of many researchers for its remarkable antiviral, antitumor and antidepressant effects. AIM OF THE STUDY: To find plant molecules that inhibit the alkaline nuclease (AN) of herpes simplex virus type 1 (HSV-1) and suppress viral replication. MATERIALS AND METHODS: Bioinformatics methods were used to determine which compounds from a variety of natural compounds in our laboratory interact with AN. By this means we predicted that hypericin may interact with AN and suppress HSV-1 replication. Experiments were then carried out to verify whether hypericin inhibits the bioactivity of AN. The Pichia pastoris expression system was used to obtain recombinant AN. The exonuclease and endonuclease activity of AN treated with hypericin were tested by electrophoresis. Immunohistochemical staining of the HSV-1 nucleocapsids was used to find out whether hypericin inhibits the intracellular function of AN. Real-time PCR and western blotting analysis were performed to test viral gene expression and viral protein synthesis. The extent of viral replication inhibited by hypericin was determined by a plaque assay and a time of addition assay. RESULTS: Recombinant AN was obtained by Pichia pastoris expression system. The exonuclease and endonuclease activity of recombinant AN were inhibited by hypericin in the electrophoresis assay. Hypericin showed no inhibitory effect on BeyoZonase™ Super Nuclease or DNase I. T5 Exonuclease activity was inhibited partially by10 µM hypericin, and was completely suppressed by 50 µM hypericin. Hind Ⅲ was inhibited by hypericin at concentrations greater than 100 µM, but EcoR I, BamH I, and Sal I were not inhibited by hypericin. HSV-1 nucleocapsids gathered in the nucleus when the viruses were treated with hypericin. Plaque formation was significantly reduced by hypericin (EC50 against HSV-1 F is 2.59 ± 0.08 µM and EC50 against HSV-1 SM44 is 2.94 ± 0.10 µM). UL12, ICP27, ICP8, gD, and UL53 gene expression (P < 0.01, 4.0 µM hypericin treated group vs control group) and ICP4 (P < 0.05, 6.0 µM hypericin treated group vs control group), ICP8 and gD (P < 0.05, 2.0 µM hypericin treated group vs control group) protein synthesis were inhibited by hypericin. In the time of addition assay, HSV-1 was suppressed by hypericin in the early stages of viral replication. Hypericin exhibits potent virucidal activity against HSV-1 and inhibits the adsorption and penetration of HSV-1. CONCLUSION: Hypericin inhibits the bioactivity of AN and suppresses HSV-1 replication. The data revealed a novel mechanism of the antiherpetic effect of hypericin.

ISG20 inhibits bluetongue virus replication.

ISG20 is an interferon-inducible exonuclease that inhibits virus replication. Although ISG20 is thought to degrade viral RNA, the antiviral mechanism and specificity of ISG20 remain unclear. In this study, the antiviral role of ovine ISG20 (oISG20) in bluetongue virus â€‹(BTV) infection was investigated. It was found that BTV infection up-regulated the transcription of ovine ISG20 (oISG20) in a time- and BTV multiplicity of infection (MOI)-dependent manner. Overexpression of oISG20 suppressed the production of BTV genome, proteins, and virus titer, whereas the knockdown of oISG20 increased viral replication. oISG20 was found to co-localize with BTV proteins VP4, VP5, VP6, and NS2, but only directly interacted with VP4. Exonuclease defective oISG20 significantly decreased the inhibitory effect on BTV replication. In addition, the interaction of mutant oISG20 and VP4 was weakened, suggesting that binding to VP4 was associated with the inhibition of BTV replication. The present data characterized the anti-BTV effect of oISG20, and provides a novel clue for further exploring the inhibition mechanism of double-stranded RNA virus by ISG20.

A single point mutation in the Plasmodium falciparum 3'-5' exonuclease does not alter piperaquine susceptibility.

BACKGROUND: The rise in Plasmodium falciparum resistance to dihydroartemisinin-piperaquine (DHA-PPQ) treatment has been documented in the Greater Mekong Subregion with associations with mutations in the P. falciparum chloroquine resistance transporter (pfcrt) and plasmepsin 2 (pfpm2) genes. However, it is unclear whether other genes also play a role with PPQ resistance, such as the E415G mutation in the exonuclease (pfexo) gene. The aim of this study was to investigate the role of this mutation in PPQ resistance by generating transgenic parasites expressing the pfexo-E415G mutant allele. METHODS: Transgenic parasite clones carrying the E415G mutation in PfEXO of the B5 isolate were derived by CRISPR-Cas9 gene editing and verified using PCR and gene sequencing. Polymorphisms of pfkelch-13, pfcrt, and pfexo were examined by PCR while the copy number variations of pfpm2 were examined by both relative quantitative real-time PCR and the duplication breakpoint assay. Drug sensitivity against a panel of antimalarials, the ring-stage survival assay (RSA), the PPQ survival assay (PSA), and bimodal dose-response curves were used to evaluate antimalarial susceptibility. RESULTS: The transgenic line, B5-rexo-E415G-B8, was successfully generated. The PPQ-IC90, %PPQ survival, and the bimodal dose-response clearly showed that E415G mutation in PfEXO of B5 isolate remained fully susceptible to PPQ. Furthermore, growth assays demonstrated that the engineered parasites grew slightly faster than the unmodified parental isolates whereas P. falciparum isolates harbouring pfkelch-13, pfcrt, and pfexo mutations with multiple copies of pfpm2 grew much more slowly. CONCLUSIONS: Insertion of the E415G mutation in PfEXO did not lead to increased PPQ-IC90 and %PPQ survival, suggesting that this mutation alone may not be associated with PPQ resistance, but could still be an important marker if used in conjunction with other markers for monitoring PPQ-resistant parasites. The results also highlight the importance of monitoring and evaluating suspected genetic mutations with regard to parasite fitness and resistance.

Two-Metal Ion-Dependent Enzymes as Potential Antiviral Targets in Human Herpesviruses.

The majority of drug discovery efforts against herpesviruses have focused on nucleoside analogs that target viral DNA polymerases, agents that are associated with dose-limiting toxicity and/or a narrow spectrum of activity. We are pursuing a strategy based on targeting two-metal ion-dependent (TMID) viral enzymes. This family of enzymes consists of structurally related proteins that share common active sites containing conserved carboxylates predicted to coordinate divalent cations essential for catalysis. Compounds that target TMID enzymes, such as HIV integrase and influenza endoribonuclease, have been successfully developed for clinical use. HIV integrase inhibitors have been reported to inhibit replication of herpes simplex virus (HSV) and other herpesviruses; however, the molecular targets of their antiviral activities have not been identified. We employed a candidate-based approach utilizing several two-metal-directed chemotypes and the potential viral TMID enzymatic targets in an effort to correlate target-based activity with antiviral potency. The panel of compounds tested included integrase inhibitors, the anti-influenza agent baloxavir, three natural products previously shown to exhibit anti-HSV activity, and two 8-hydroxyquinolines (8-HQs), AK-157 and AK-166, from our in-house program. The integrase inhibitors exhibited weak overall anti-HSV-1 activity, while the 8-HQs were shown to inhibit both HSV-1 and cytomegalovirus (CMV). Target-based analysis demonstrated that none of the antiviral compounds acted by inhibiting ICP8, contradicting previous reports. On the other hand, baloxavir inhibited the proofreading exonuclease of HSV polymerase, while AK-157 and AK-166 inhibited the alkaline exonuclease UL12. In addition, AK-157 also inhibited the catalytic activity of the HSV polymerase, which provides an opportunity to potentially develop dual-targeting agents against herpesviruses. IMPORTANCE Human herpesviruses (HHVs) establish lifelong latent infections, which undergo periodic reactivation and remain a major cause of morbidity and mortality, especially in immunocompromised individuals. Currently, HHV infections are treated primarily with agents that target viral DNA polymerase, including nucleoside analogs; however, long-term treatment can be complicated by the development of drug resistance. New therapies with novel modes of action would be important not only for the treatment of resistant viruses but also for use in combination therapy to reduce dose-limiting toxicities and potentially eliminate infection. Since many essential HHV proteins are well conserved, inhibitors of novel targets would ideally exhibit broad-spectrum activity against multiple HHVs.

Human Antimicrobial RNases Inhibit Intracellular Bacterial Growth and Induce Autophagy in Mycobacteria-Infected Macrophages.

The development of novel treatment against tuberculosis is a priority global health challenge. Antimicrobial proteins and peptides offer a multifaceted mechanism suitable to fight bacterial resistance. Within the RNaseA superfamily there is a group of highly cationic proteins secreted by innate immune cells with anti-infective and immune-regulatory properties. In this work, we have tested the human canonical members of the RNase family using a spot-culture growth inhibition assay based mycobacteria-infected macrophage model for evaluating their anti-tubercular properties. Out of the seven tested recombinant human RNases, we have identified two members, RNase3 and RNase6, which were highly effective against Mycobacterium aurum extra- and intracellularly and induced an autophagy process. We observed the proteins internalization within macrophages and their capacity to eradicate the intracellular mycobacterial infection at a low micro-molar range. Contribution of the enzymatic activity was discarded by site-directed mutagenesis at the RNase catalytic site. The protein induction of autophagy was analyzed by RT-qPCR, western blot, immunofluorescence, and electron microscopy. Specific blockage of auto-phagosome formation and maturation reduced the protein's ability to eradicate the infection. In addition, we found that the M. aurum infection of human THP1 macrophages modulates the expression of endogenous RNase3 and RNase6, suggesting a function in vivo. Overall, our data anticipate a biological role for human antimicrobial RNases in host response to mycobacterial infections and set the basis for the design of novel anti-tubercular drugs.

Interferon-Stimulated Gene (ISG)-Expression Screening Reveals the Specific Antibunyaviral Activity of ISG20.

Bunyaviruses pose a significant threat to human health, prosperity, and food security. In response to viral infections, interferons (IFNs) upregulate the expression of hundreds of interferon-stimulated genes (ISGs), whose cumulative action can potently inhibit the replication of bunyaviruses. We used a flow cytometry-based method to screen the ability of ∼500 unique ISGs from humans and rhesus macaques to inhibit the replication of Bunyamwera orthobunyavirus (BUNV), the prototype of both the Peribunyaviridae family and the Bunyavirales order. Candidates possessing antibunyaviral activity were further examined using a panel of divergent bunyaviruses. Interestingly, one candidate, ISG20, exhibited potent antibunyaviral activity against most viruses examined from the Peribunyaviridae, Hantaviridae, and Nairoviridae families, whereas phleboviruses (Phenuiviridae) largely escaped inhibition. Similar to the case against other viruses known to be targeted by ISG20, the antibunyaviral activity of ISG20 is dependent upon its functional RNase activity. Through use of an infectious virus-like particle (VLP) assay (based on the BUNV minigenome system), we confirmed that gene expression from all 3 viral segments is strongly inhibited by ISG20. Using in vitro evolution, we generated a substantially ISG20-resistant BUNV and mapped the determinants of ISG20 sensitivity/resistance. Taking all the data together, we report that ISG20 is a broad and potent antibunyaviral factor but that some bunyaviruses are remarkably ISG20 resistant. Thus, ISG20 sensitivity/resistance may influence the pathogenesis of bunyaviruses, many of which are emerging viruses of clinical or veterinary significance.IMPORTANCE There are hundreds of bunyaviruses, many of which cause life-threatening acute diseases in humans and livestock. The interferon (IFN) system is a key component of innate immunity, and type I IFNs limit bunyaviral propagation both in vitro and in vivo Type I IFN signaling results in the upregulation of hundreds of IFN-stimulated genes (ISGs), whose concerted action generates an "antiviral state." Although IFNs are critical in limiting bunyaviral replication and pathogenesis, much is still unknown about which ISGs inhibit bunyaviruses. Using ISG-expression screening, we examined the ability of ∼500 unique ISGs to inhibit Bunyamwera orthobunyavirus (BUNV), the prototypical bunyavirus. Using this approach, we identified ISG20, an interferon-stimulated exonuclease, as a potent inhibitor of BUNV. Interestingly, ISG20 possesses highly selective antibunyaviral activity, with multiple bunyaviruses being potently inhibited while some largely escape inhibition. We speculate that the ability of some bunyaviruses to escape ISG20 may influence their pathogenesis.

Interferon-inducible ribonuclease ISG20 inhibits hepatitis B virus replication through directly binding to the epsilon stem-loop structure of viral RNA.

Hepatitis B virus (HBV) replicates its DNA genome through reverse transcription of a viral RNA pregenome. We report herein that the interferon (IFN) stimulated exoribonuclease gene of 20 KD (ISG20) inhibits HBV replication through degradation of HBV RNA. ISG20 expression was observed at basal level and was highly upregulated upon IFN treatment in hepatocytes, and knock down of ISG20 resulted in elevation of HBV replication and attenuation of IFN-mediated antiviral effect. The sequence element conferring the susceptibility of HBV RNA to ISG20-mediated RNA degradation was mapped at the HBV RNA terminal redundant region containing epsilon (ε) stem-loop. Furthermore, ISG20-induced HBV RNA degradation relies on its ribonuclease activity, as the enzymatic inactive form ISG20D94G was unable to promote HBV RNA decay. Interestingly, ISG20D94G retained antiviral activity against HBV DNA replication by preventing pgRNA encapsidation, resulting from a consequence of ISG20-ε interaction. This interaction was further characterized by in vitro electrophoretic mobility shift assay (EMSA) and ISG20 was able to bind HBV ε directly in absence of any other cellular proteins, indicating a direct ε RNA binding capability of ISG20; however, cofactor(s) may be required for ISG20 to efficiently degrade ε. In addition, the lower stem portion of ε is the major ISG20 binding site, and the removal of 4 base pairs from the bottom portion of ε abrogated the sensitivity of HBV RNA to ISG20, suggesting that the specificity of ISG20-ε interaction relies on both RNA structure and sequence. Furthermore, the C-terminal Exonuclease III (ExoIII) domain of ISG20 was determined to be responsible for interacting with ε, as the deletion of ExoIII abolished in vitro ISG20-ε binding and intracellular HBV RNA degradation. Taken together, our study sheds light on the underlying mechanisms of IFN-mediated HBV inhibition and the antiviral mechanism of ISG20 in general.

Topical application of a film-forming emulgel dressing that controls the release of stratifin and acetylsalicylic acid and improves/prevents hypertrophic scarring.

Here, we evaluate the efficacy of an emulgel dressing to control the release of an antifibrogenic factor, stratifin (SFN), along with an anti-inflammatory drug, acetylsalicylic acid (ASA), to be used as a wound dressing with hypertrophic scar reducing features. Emulgel dressings were prepared by dispersing positively charged submicron vesicles in carboxymethyl cellulose gel. Release kinetics of SFN/ASA and toxicity for primary skin cells were assessed in vitro. Antifibrogenic efficacy of medicated emulgel dressings was tested on a rabbit ear fibrotic model. Following topical application on the wounds, emulgels formed an occlusive film and controlled the release of SFN and ASA for 7 and 24 hours, respectively. Wounds treated with SFN/ASA-containing emulgel dressings showed an 80% reduction in scar elevation compared with untreated controls. Topical formulations were nontoxic for cultured human keratinocytes and fibroblasts. Inflammation was significantly controlled in treated wounds, as shown by a reduced number of infiltrated CD3(+) T cells (p < 0.001) and macrophages. SFN/ASA-treated wounds showed a significantly higher (p < 0.001) expression of matrix metalloproteinase-1, resulting in reduced collagen deposition and less scarring. Film-forming emulgel dressings that control the release of antifibrogenic and anti-inflammatory factors provide an excellent treatment option for postburn hypertrophic scar management.

Localized controlled release of stratifin reduces implantation-induced dermal fibrosis.

Localized controlled release of anti-fibrogenic factors can potentially prevent tissue fibrosis surrounding biomedical prostheses, such as vascular stents and breast implants. We have previously demonstrated that therapeutic intervention with topically applied stratifin in a rabbit ear fibrotic model not only prevents dermal fibrosis but also promotes more normal tissue repair by regulating extracellular matrix deposition. In this work, the anti-fibrogenic effect of a controlled release form of stratifin was investigated in the prevention of fibrosis induced by dermal poly(lactic-co-glycolic acid) (PLGA) microsphere/poly(vinyl alcohol) (PVA) hydrogel implants. Pharmacodynamic effects were evaluated by histopathological examination of subcutaneous tissue surrounding implanted composites. Controlled release of stratifin from PLGA microsphere/PVA hydrogel implants significantly moderated dermal fibrosis and inflammation by reducing collagen deposition (30%), total tissue cellularity (48%) and infiltrated CD3(+) immune cells (81%) in the surrounding tissue compared with the stratifin-free implants. The controlled release of stratifin from implants markedly increased the level of matrix metalloproteinase-1 expression in the surrounding tissue, which resulted in less collagen deposition. These stratifin-eluting PLGA/PVA composites show promise as coatings to decrease the typical fibrosis exhibited around implanted biomedical prostheses, such as breast implants and vascular stents.

Improvement of hypertrophic scarring by using topical anti-fibrogenic/anti-inflammatory factors in a rabbit ear model.

This study investigates the scar-reducing efficacy of topical application of stratifin and acetylsalicylic acid (ASA) in a rabbit ear model. A total of five New Zealand white rabbits with four wounds per ear were examined. Either recombinant stratifin (0.002%) or ASA (0.5%) incorporated in carboxymethyl cellulose gel was topically applied on each wound at postwounding Day 5. Scars were harvested at postwounding Day 28 for histological analysis. The wounds treated with stratifin and ASA showed 82 and 73% reduction in scar volume, respectively, compared with that of untreated controls. A reduction of 57 and 41% in total tissue cellularity along with 79 and 91% reduction in infiltrated CD3+ T cells were observed in response to treatment with stratifin and ASA, respectively, compared with those of untreated controls. Wounds treated with stratifin showed a 2.8-fold increase in matrix metalloproteinase-1 expression, which resulted in a 48% decrease in collagen density compared with those of untreated controls. Qualitative wound assessment showed a reduced hypertrophic scarring in stratifin and ASA-treated wounds when compared with the controls. This study showed that topical application of either stratifin or ASA-impregnated carboxymethyl cellulose gel reduced hypertrophic scar formation following dermal injuries in a rabbit ear fibrotic model.

Composite hydrogel formulations of stratifin to control MMP-1 expression in dermal fibroblasts.

PURPOSE: Stratifin is a potent anti-fibrogenic factor that stimulates the expression of matrix metalloproteinase-1 (MMP-1) in dermal fibroblasts. The propose of this work was to develop a controlled release delivery system for stratifin that can be applied at the time of wound closure to release stratifin and stimulate the expression of MMP-1 in a sustained manner over the late stages of wound healing (after 3 days). METHODS: Stratifin was complexed to chitosan particles, which were then encapsulated in PLGA microspheres and blended into crosslinked hyaluronic acid films. In vitro release was assessed using fluorescent-tagged stratifin, cytotoxicity by MTT assay and bioactivity by measuring the levels of MMP-1 expression in cultured fibroblasts. RESULTS: The release of stratifin was delayed for 3 days and then controlled for 30 days so that 60% of the total stratifin loaded was released. The released protein significantly stimulated the expression of MMP-1 in cultured fibroblasts without compromising cell viability. By complexing to chitosan, the initial burst release was reduced, so that only 5% of stratifin was released in 3 days. CONCLUSION: This stratifin delivery system has the potential to be used as an anti-fibrogenic factor-associated wound insert for improving post-surgical scarring in closed wound.

Characterization of a novel non-specific nuclease from thermophilic bacteriophage GBSV1.

BACKGROUND: Thermostable enzymes from thermophiles have attracted extensive studies. In this investigation, a nuclease-encoding gene (designated as GBSV1-NSN) was obtained from a thermophilic bacteriophage GBSV1 for the first time. RESULTS: After recombinant expression in Escherichia coli, the purified GBSV1-NSN exhibited non-specific nuclease activity, being able to degrade various nucleic acids, including RNA, single-stranded DNA and double-stranded DNA that was circular or linear. Based on sequence analysis, the nuclease shared no homology with any known nucleases, suggesting that it was a novel nuclease. The characterization of the recombinant GBSV1-NSN showed that its optimal temperature and pH were 60 degrees C and 7.5, respectively. The results indicated that the enzymatic activity was inhibited by enzyme inhibitors or detergents, such as ethylene diamine tetraacetic acid, citrate, dithiothreitol, beta-mercaptoethanol, guanidine hydrochloride, urea and SDS. In contrast, the nuclease activity was enhanced by TritonX-100, Tween-20 or chaps to approximately 124.5% - 141.6%. The Km of GBSV1-NSN nuclease was 231, 61 and 92 microM, while its kcat was 1278, 241 and 300 s-1 for the cleavage of dsDNA, ssDNA and RNA, respectively. CONCLUSION: Our study, therefore, presented a novel thermostable non-specific nuclease from thermophilic bacteriophage and its overexpression and purification for scientific research and applications.

[Effect of 14-3-3sigma on transcriptional activity of p73 gene].

BACKGROUND & OBJECTIVE: p53 gene is the main regulator of 14-3-3sigma. Activated p53 could induce the expression of 14-3-3sigma, while 14-3-3sigma stabilizes the expression of p53 and enhances its transcriptional activity. p63 and p73, the members of p53 family, also have some functions similar to p53. This study was to investigate the effect of 14-3-3sigma on the transcriptional activity of p73. METHODS: Luciferase reporter assay, reverse transcription-polymerase chain reaction (RT-PCR), and Western blot were used to evaluate the effect of 14-3-3sigma on the transcriptional activity of p73 in p53-deficient human lung carcinoma cell line H1299. Colony formation test was used to evaluate the effect of 14-3-3sigma on the transcriptional activity of p73 in p53-mutant human breast cancer cell line MDA-MB-436. RESULTS: The luciferase activities induced by bax and p21WAF1 promotors were significantly higher in p73-transfected H1299 cells than in control H1299 cells (P<0.01), and were further increased by the transfection of p73 (25 ng) and 14-3-3sigma (100, 200, and 400 ng) in a dose-dependent manner (P<0.01). The expression of bax and p21WAF1 were higher in p73-transfected H1299 cells than in control H1299 cells, and were significantly higher in p73-and 14-3-3sigma-transfected H1299 cells than in p73-transfected H1299 cells (P<0.01). The number of colonies was fewer in p73-transfected MDA-MB-436 cells than in control MDA-MB-436 cells, and the colonies were significantly smaller in p73-and 14-3-3sigma-transfected H1299 cells than in p73-transfected H1299 cells (P<0.01). CONCLUSION: 14-3-3sigma can enhance the transcriptional activity of p73 in a dose-dependent manner.

Stratifin-induced matrix metalloproteinase-1 in fibroblast is mediated by c-fos and p38 mitogen-activated protein kinase activation.

Previously, we have demonstrated that keratinocyte releasable stratifin, also known as 14-3-3 sigma protein, stimulates matrix metalloproteinase (MMP)-1 expression in dermal fibroblasts. In this study, we showed that stratifin induced fibroblast MMP-1 messenger ribonucleic acid (mRNA) and protein levels through p38 mitogen-activated protein kinase (MAPK). Our data indicated that treatment of dermal fibroblasts with stratifin resulted in rapid and transient upregulation of c-jun and c-fos mRNA levels. We also demonstrated that SB203580 (SB), a specific inhibitor of p38 MAPK activity, inhibited the activation of fibroblast MMP-1 mRNA expression by stratifin. Subsequently, western blot analysis revealed phosphorylation of p38 at 90 min after stratifin stimulation and this was decreased to approximately 50% of the maximum value by 120 min. Stratifin was demonstrated to increase MMP-1 protein levels starting at 4 h and reaching its peak at 12-24 h. Furthermore, SB significantly blocked the stratifin induction of MMP-1 protein levels (***p<0.005, n=3). Microarray analysis of stratifin-treated fibroblasts shows an increase in Elk4/Sap1 mRNA expression and this finding was confirmed by northern blot analysis. Our results indicate that stratifin markedly increase Elk4/Sap1 mRNA expression in a time-dependent fashion. In conclusion, stratifin stimulates fibroblast MMP-1 levels through the activation of c-fos and MAPK pathway.

Differentiated keratinocyte-releasable stratifin (14-3-3 sigma) stimulates MMP-1 expression in dermal fibroblasts.

Through the use of a keratinocyte/fibroblast co-culture system, we have recently identified a potent keratinocyte-derived anti-fibrogenic factor (KDAF) for dermal fibroblasts. A sequential chromatography of the active fractions of keratinocyte-conditioned medium (KCM) and peptide mapping of the candidate proteins identified KDAF as being the keratinocyte-releasable 14-3-3 sigma (14-3-3sigma) protein, which is also known as stratifin. In this study, we hypothesize that differentiated, but not proliferating, keratinocytes are the primary source of releasable 14-3-3sigma in conditioned medium. To address this hypothesis, in a longitudinal study, keratinocyte differentiation was induced by growing these cells in a medium consisting of 50% keratinocyte serum-free medium (KSFM) and 50% Dulbecco's modified eagle's medium without any additives for up to 20 d. When KCM was collected every other day and added to fibroblasts, the level of matrix metalloproteinase (MMP)-1 mRNA expression was markedly increased in fibroblasts receiving KCM and this increase was even greater in cells receiving conditioned media collected at later time points relative to that of controls. The results of a western blot analysis further showed a marked increase in the expression of 14-3-3sigma protein in keratinocytes grown in test medium from day 4 to day 10. This finding was consistent with the levels of 14-3-3sigma mRNA expression in differentiated keratinocytes. In contrast to a very high level of 14-3-3sigma mRNA expression seen in keratinocytes, fibroblasts that are highly responsive to14-3-3sigma were unable to express this factor. Interestingly, the level of 14-3-3sigma mRNA expression was markedly higher in keratinocytes co-cultured with fibroblasts relative to that of mono-cultured keratinocytes. In conclusion, this study provides evidence that keratinocytes express a high level of 14-3-3sigma at the levels of mRNA and protein. But the releasable form of 14-3-3sigma protein was only found in conditioned medium derived from differentiated keratinocytes. Further, our recently purified recombinant 14-3-3sigma protein mimics the collagenase stimulatory effect of KCM in dermal fibroblasts.

Resistance towards exonucleases of dinucleotides with stereochemically altered internucleotide phosphate bonds.

Kinetic constants for the hydrolytic susceptibility of the internucleotide phosphate bond in normal dinucleotides [e.g., 2'-deoxycytidylyl-(3'>5')-2'-deoxyuridine (dCpdU) and 2'-deoxyadenylyl-(3'-->5')-2'-deoxycytidine (dApdC)] and isomeric dinucleotides [e.g., 2'-deoxycytidylyl-(3'-->5')-1'-deoxy-2'-isouridine (dCpisodU) and 1'-deoxy-2'-isoadenylyl-(3'-->5')-2'-deoxycytidine (isodApdC)], toward 5'- and 3'-exonucleases, phosphodiesterase I (PDE I) and phosphodiesterase II (PDE II) were experimentally determined and remarkable differences emerged. The study is of importance in the discovery of nuclease-stable inhibitors of HIV integrase, but may also have ramifications in the area of anti-sense oligonucleotides of therapeutic interest.

Insulin suppresses collagenase stimulatory effect of stratifin in dermal fibroblasts.

A delicate balance between synthesis and degradation of extracellular matrix (ECM) by matrix metalloproteinases (MMPs) is an essential feature of tissue remodeling. We have recently demonstrated that keratinocyte releasable stratifin, also known as 14-3-3 sigma protein, plays a critical role in modulating collagenase (MMP-1) mRNA expression in human dermal fibroblasts. In this study, we further characterized the collagenase stimulatory effect of stratifin in dermal fibroblasts and evaluated its effect in the presence and absence of insulin. Our data indicate that stratifin increases the expression of collagenase mRNA more than 20-fold in dermal fibroblasts, grown in either Dulbecco's modified Eagle's medium (DMEM) plus 2% or 10% fetal bovine serum (FBS). Collagenase stimulatory effect of stratifin was completely blocked, when fibroblasts were cultured in test medium consisting of 50% keratinocyte serum-free medium (KSFM) and 50% DMEM. The collagenase suppressive effect of test medium was directly proportional to the volume of KSFM used. As this medium contained insulin, we then evaluated the collagenase stimulatory effect of stratifin in dermal fibroblasts in the presence and absence of insulin. The results revealed that stratifin significantly increased the expression of collagenase mRNA/18S (*p < 0.05, n = 3) ratio, while insulin significantly decreased the expression of collagenase mRNA/18S (*p < 0.05, n = 3) ratio. The insulin inhibitory effect on collagenase mRNA expression was time and dose dependent. The maximal inhibitory effect of insulin was seen at 36 h post treatment. In conclusion, stratifin stimulates the expression of collagenase mRNA expression in dermal fibroblasts and this effect is suppressed by insulin treatment.

Antisense oligonuclotides with oxetane-constrained cytidine enhance heteroduplex stability, and elicit satisfactory RNase H response as well as showing improved resistance to both exo and endonucleases.

Antisense oligonucleotides (AONs) with single and double oxetane C modifications [1',2'-oxetane constrained cytidine, 1-(1',3'-O-anhydro-beta-D-psicofuranosyl)cytosine] have been evaluated, in comparison with the corresponding T-modified AONs, for their antisense potentials by targeting to a 15mer complementary RNA. Although the C modified mixmer AONs show approximately 3 degrees C drop per modification in melting temperature (Tm) of their hybrid AON-RNA duplexes, they are found to be good substrates for RNase H, in comparison with the native AON-RNA duplex. An AON with double C modifications along with 3'-DPPZ (dipyridophenazine) conjugation shows the Tm of the hybrid duplexes as high as that of the native, and the RNase H activity as good as its unconjugated counterpart. A detailed Michaelis-Menten kinetic analysis of RNase H cleavage showed that the single and double C modified AON-RNA duplexes as well as double C modifications along with 3'-DPPZ have catalytic activities (kcat) close to the native. However, the R Nase H binding affinity (1/Km) showed a slight decrease with increase in the number of modifications, which results in less effective enzyme activity (kcat/Km) for C modified AON-RNA duplexes. All oxetane modified AON-RNA hybrids showed a correlation of Tm with the 1/Km, Vmax, or Vmax/Km. The C modified AONs (with 3'-DPPZ), as in the T counterpart, showed an enhanced tolerance towards the endonuclease and exonuclease degradation compared to the native (the oxetane-sugar and the DPPZ based AONs are non-toxic to K562 cell growth, ref. 18). Thus a balance has been found between exo and endonuclease stability vis-a-vis thermostability of the heteroduplex and the R Nase H recruitment capability and cleavage with the oxetane-constrained cytidine incorporated AONs as potential antisense candidates with a fully phosphate backbone for further biological assessment.

Excision of 8-methylguanine site-specifically incorporated into oligonucleotide substrates by the AlkA protein of Escherichia coli.

8-Methyl-2'-deoxyguanosine (8-medGuo) has been shown to be a major stable alkylation product of 2'-deoxyguanosine induced by methyl radical attack on DNA. Moreover, by using primer extension assays, the latter DNA modification has recently been reported to be a miscoding lesion by generating G to C and G to T transversions and deletions in vitro. However, no data have been reported up to now, concerning the processing of this C8-alkylated nucleoside by the DNA repair machinery. Therefore, we have investigated the capability of excision of 8-methylguanine (8-meGua) site specifically incorporated into oligonucleotide substrates by several bacterial, yeast and mammalian DNA N-glycosylases. The results show that the 3-methyladenine (3-meAde) DNA glycosylase II (AlkA protein) from Escherichia coli is the only DNA N-glycosylase tested able to remove 8-meGua from double-stranded DNA fragments. Moreover, the activity of AlkA for 8-meGua varied markedly depending on the opposite base in DNA, being the highest with Adenine and Thymine and the lowest with Cytosine and Guanine. The removal of 8-meGua by AlkA protein was compared to that of 7-methylguanine (7-meGua) and hypoxanthine (Hx). The rank of damage as a substrate for AlkA being 7-meGua>8-meGua>Hx. In contrast, the human 3-meAde DNA N-glycosylase (Mpg) is not able to release 8-meGua paired with any of the four DNA bases. We also show that, DNA N-glycosylases involved in the removal of oxidative damage, such as Fpg or Nth proteins from E. coli, Ntg1, Ntg2 or Ogg1 proteins of Saccharomyces cerevisiae, or human Ogg1 do not release 8-meGua placed opposite any of the four DNA bases. Furthermore, HeLa and Chinese hamster ovary (CHO) cell free protein extracts do not show any cleavage activity at 8-meGua paired with adenine or cytosine, which suggests the absence of base excision repair (BER) of this lesion in mammalian cells.