Oncolytic HSV-1 G207 Immunovirotherapy for Pediatric High-Grade Gliomas.

BACKGROUND: Outcomes in children and adolescents with recurrent or progressive high-grade glioma are poor, with a historical median overall survival of 5.6 months. Pediatric high-grade gliomas are largely immunologically silent or "cold," with few tumor-infiltrating lymphocytes. Preclinically, pediatric brain tumors are highly sensitive to oncolytic virotherapy with genetically engineered herpes simplex virus type 1 (HSV-1) G207, which lacks genes essential for replication in normal brain tissue. METHODS: We conducted a phase 1 trial of G207, which used a 3+3 design with four dose cohorts of children and adolescents with biopsy-confirmed recurrent or progressive supratentorial brain tumors. Patients underwent stereotactic placement of up to four intratumoral catheters. The following day, they received G207 (107 or 108 plaque-forming units) by controlled-rate infusion over a period of 6 hours. Cohorts 3 and 4 received radiation (5 Gy) to the gross tumor volume within 24 hours after G207 administration. Viral shedding from saliva, conjunctiva, and blood was assessed by culture and polymerase-chain-reaction assay. Matched pre- and post-treatment tissue samples were examined for tumor-infiltrating lymphocytes by immunohistologic analysis. RESULTS: Twelve patients 7 to 18 years of age with high-grade glioma received G207. No dose-limiting toxic effects or serious adverse events were attributed to G207 by the investigators. Twenty grade 1 adverse events were possibly related to G207. No virus shedding was detected. Radiographic, neuropathological, or clinical responses were seen in 11 patients. The median overall survival was 12.2 months (95% confidence interval, 8.0 to 16.4); as of June 5, 2020, a total of 4 of 11 patients were still alive 18 months after G207 treatment. G207 markedly increased the number of tumor-infiltrating lymphocytes. CONCLUSIONS: Intratumoral G207 alone and with radiation had an acceptable adverse-event profile with evidence of responses in patients with recurrent or progressive pediatric high-grade glioma. G207 converted immunologically "cold" tumors to "hot." (Supported by the Food and Drug Administration and others; ClinicalTrials.gov number, NCT02457845.).

Attachment and Postattachment Receptors Important for Hepatitis C Virus Infection and Cell-to-Cell Transmission.

Hepatitis C virus (HCV) requires multiple receptors for its attachment to and entry into cells. Our previous studies found that human syndecan-1 (SDC-1), SDC-2, and T cell immunoglobulin and mucin domain-containing protein 1 (TIM-1) are HCV attachment receptors. Other cell surface molecules, such as CD81, Claudin-1 (CLDN1), Occludin (OCLN), SR-BI, and low-density lipoprotein receptor (LDLR), function mainly at postattachment steps and are considered postattachment receptors. The underlying molecular mechanisms of different receptors in HCV cell-free and cell-to-cell transmission remain elusive. In the present study, we used a clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 technology, gene-specific small interfering RNAs, and a newly developed luciferase-based reporter system to quantitatively determine the importance of individual receptors in HCV cell-free and cell-to-cell transmission. Knockouts of SDC-1 and SDC-2 resulted in remarkable reductions of HCV infection and cell attachment, whereas SDC-3 and SDC-4 knockouts did not affect HCV infection. Defective HCV attachment to SDC-1 and/or SDC-2 knockout cells was completely restored by SDC-1 and SDC-2 but not SDC-4 expression. Knockout of the attachment receptors SDC-1, SDC-2, and TIM-1 also modestly decreased HCV cell-to-cell transmission. In contrast, silencing and knockout of the postattachment receptors CD81, CLDN1, OCLN, SR-BI, and LDLR greatly impaired both HCV cell-free and cell-to-cell transmission. Additionally, apolipoprotein E was found to be important for HCV cell-to-cell spread, but very-low-density lipoprotein (VLDL)-containing mouse serum did not affect HCV cell-to-cell transmission, although it inhibited cell-free infection. These findings demonstrate that attachment receptors are essential for initial HCV binding and that postattachment receptors are important for both HCV cell-free and cell-to-cell transmission.IMPORTANCE The importance and underlying molecular mechanisms of cell surface receptors in HCV cell-free and cell-to-cell transmission are poorly understood. The role of some of the HCV attachment and postattachment receptors in HCV infection and cell-to-cell spread remains controversial. Using CRISPR-Cas9-mediated knockouts of specific cellular genes, we demonstrate that both SDC-1 and SDC-2, but not SDC-3 or SDC-4, are bona fide HCV attachment receptors. We also used a newly developed luciferase-based reporter system to quantitatively determine the importance of attachment and postattachment receptors in HCV cell-to-cell transmission. SDC-1, SDC-2, TIM-1, and SR-BI were found to modestly promote HCV cell-to-cell spread. CD81, CLDN1, OCLN, and LDLR play more important roles in HCV cell-to-cell transmission. Likewise, apolipoprotein E (apoE) is critically important for HCV cell-to-cell spread, unlike VLDL-containing mouse serum, which did not affect HCV cell-to-cell spread. These findings suggest that the mechanism(s) of HCV cell-to-cell spread differs from that of cell-free infection.

The Serum Very-Low-Density Lipoprotein Serves as a Restriction Factor against Hepatitis C Virus Infection.

UNLABELLED: Recent studies demonstrated that transgenic mice expressing key human hepatitis C virus (HCV) receptors are susceptible to HCV infection, albeit at very low efficiency. Robust mouse models of HCV infection and replication are needed to determine the importance of host factors in HCV replication, pathogenesis, and carcinogenesis as well as to facilitate the development of antiviral agents and vaccines. The low efficiency of HCV replication in the humanized mouse models is likely due to either the lack of essential host factors or the presence of restriction factors for HCV infection and/or replication in mouse hepatocytes. To determine whether HCV infection is affected by restriction factors present in serum, we examined the effects of mouse and human sera on HCV infectivity. Strikingly, we found that mouse and human sera potently inhibited HCV infection. Mechanistic studies demonstrated that mouse serum blocked HCV cell attachment without significant effect on HCV replication. Fractionation analysis of mouse serum in conjunction with targeted mass spectrometric analysis suggested that serum very-low-density lipoprotein (VLDL) was responsible for the blockade of HCV cell attachment, as VLDL-depleted mouse serum lost HCV-inhibitory activity. Both purified mouse and human VLDL could efficiently inhibit HCV infection. Collectively, these findings suggest that serum VLDL serves as a major restriction factor of HCV infection in vivo. The results also imply that reduction or elimination of VLDL production will likely enhance HCV infection in the humanized mouse model of HCV infection and replication. IMPORTANCE: HCV is a major cause of liver diseases, such as chronic hepatitis, cirrhosis, and hepatocellular carcinoma. Recently, several studies suggested that humanized mouse or transgenic mouse expressing key HCV human receptors became susceptible to HCV infection. However, HCV infection and replication in the humanized animals were very inefficient, suggesting either the lack of cellular genes important for HCV replication or the presence of restriction factors inhibiting HCV infection and replication in the mouse. In this study, we found that both mouse and human sera effectively inhibited HCV infection. Mechanistic studies demonstrated that VLDL is the major restriction factor that blocks HCV infection. These findings suggest that VLDL is beneficial to patients by restricting HCV infection. More importantly, our findings suggest that elimination of VLDL will lead to the development of more robust mouse models for the study of HCV pathogenesis, host response to HCV infection, and evaluation of HCV vaccines.

Elucidating cellular response to treatment with viral immunotherapies in pediatric high-grade glioma and medulloblastoma.

HSV G207, a double-stranded, DNA virus, and the polio:rhinovirus chimera, PVSRIPO, a single positive-strand RNA virus, are viral immunotherapies being used to treat pediatric malignant brain tumors in clinical trials. The purpose of this work is to elucidate general response patterns and putative biomarkers of response. Multiple pediatric high-grade glioma and medulloblastoma cell lines were treated with various multiplicities of infection of G207 or PVSRIPO. There was a significant inverse correlation between expression of one HSV cellular receptor, CD111, and the lethal dose of 50% of cells (LD50) of cells treated with G207 (r = -0.985, P<0.001) but no correlation between PVSRIPO cellular receptor expression (CD155) and LD50. RNA sequencing of control cells and cells treated for 8 and 24 h revealed that there were few shared differentially expressed (DE) genes between cells treated with PVSRIPO and G207: GCLM, LANCL2, and RBM3 were enriched whilst ADAMTS1 and VEGFA were depleted. Likewise, there were few shared DE genes enriched between medulloblastoma and high-grade glioma cell lines treated with G207: GPSM2, CHECK2, SEPTIN2, EIF4G2, GCLM, GDAP1, LANCL2, and PWP1.  Treatment with G207 and PVSRIPO appear to cause disparate gene enrichment and depletion suggesting disparate molecular mechanisms in malignant pediatric brain tumors.

Combination immunotherapy with vaccine and oncolytic HSV virotherapy is time dependent.

PURPOSE: Oncolytic virotherapy or immunovirotherapy is a strategy that utilizes viruses to selectively infect and kill tumor cells while also stimulating an immune response against the tumor. Early clinical trials in both pediatric and adult patients using oncolytic herpes simplex viruses (oHSVs) have demonstrated safety and promising efficacy; however, combinatorial strategies designed to enhance oncolysis while also promoting durable T cell responses for sustaining disease remission are likely required. We hypothesized that combining the direct tumor cell killing and innate immune stimulation by oHSV with a vaccine that promotes T cell mediated immunity may lead to more durable tumor regression. EXPERIMENTAL DESIGN: To this end, we investigated the preclinical efficacy and potential synergy of combining oHSV with a self-assembling nanoparticle vaccine co-delivering peptide antigens and Toll-like receptor-7 and -8 agonists (TLR-7/8a) (referred to as SNAPvax™), that induces robust tumor specific T cell immunity. We then assessed how timing of the treatments (i.e., vaccine before or after oHSV) impacts T cell responses, viral replication, and preclinical efficacy. RESULTS: The sequence of treatments was critical, as survival was significantly enhanced when the SNAPvax™ vaccine was given prior to oHSV. Increased clinical efficacy was associated with reduced tumour volume and increases in virus replication and tumor antigen specific CD8+ T cells. CONCLUSIONS: These findings substantiate the criticality of combination immunotherapy timing and provide preclinical support for combining SNAPvax with oHSV as a promising treatment approach for both pediatric and adult tumors.

CHD2 Regulates Neuron-glioma Interactions in Pediatric Glioma.

High-grade gliomas (HGG) are deadly diseases for both adult and pediatric patients. Recently, it has been shown that neuronal activity promotes progression of multiple subgroups of HGG. However, epigenetic mechanisms that govern this process remain elusive. Here we report that the chromatin remodeler CHD2 regulates neuron-glioma interactions in diffuse midline glioma (DMG) characterized by onco-histone H3.1K27M. Depletion of CHD2 in H3.1K27M DMG cells compromises cell viability and neuron-to-glioma synaptic connections in vitro, neuron-induced proliferation of H3.1K27M DMG cells in vitro and in vivo, activity-dependent calcium transients in vivo, and extends the survival of H3.1K27M DMG-bearing mice. Mechanistically, CHD2 coordinates with the transcription factor FOSL1 to control the expression of axon-guidance and synaptic genes in H3.1K27M DMG cells. Together, our study reveals a mechanism whereby CHD2 controls the intrinsic gene program of the H3.1K27M DMG subtype, which in turn regulates the tumor growth-promoting interactions of glioma cells with neurons.

Intraventricular immunovirotherapy; a translational step forward.

Oncolytic virotherapy with intratumoral engineered type-1 herpes simplex virus (HSV) has been proven safe with promising efficacy in recent clinical trials for treatment of both pediatric and adult high-grade glioma. However, this approach excludes patients with tumors in surgically inaccessible and/or eloquent brain regions. Current delivery methods are also unable to access/treat those patients with metastatic disease in the spinal cord and/or leptomeningeal disease. A recent preclinical study has paved the way for clinical translation of intraventricular administration of oHSV by identifying and mitigating the toxicity associated with this route for therapeutic benefit in murine models of disseminated medulloblastoma. This work may ultimately allow for targeting of intractable disease and provides a feasible option for the repetitive dosing of clinically relevant immunovirotherapy, G207.

Clinical advances in oncolytic virotherapy for pediatric brain tumors.

Malignant brain tumors constitute nearly one-third of cancer diagnoses in children and have recently surpassed hematologic malignancies as the most lethal neoplasm in the pediatric population. Outcomes for children with brain tumors are unacceptably poor and current standards of care-surgical resection, chemotherapy, and radiation-are associated with significant long-term morbidity. Oncolytic virotherapy has emerged as a promising immunotherapy for the treatment of brain tumors. While the majority of brain tumor clinical trials utilizing oncolytic virotherapy have been in adults, five viruses are being tested in pediatric brain tumor clinical trials: herpes simplex virus (G207), reovirus (pelareorep/Reolysin), measles virus (MV-NIS), poliovirus (PVSRIPO), and adenovirus (DNX-2401, AloCELYVIR). Herein, we review past and current pediatric immunovirotherapy brain tumor trials including the relevant preclinical and clinical research that contributed to their development. We describe mechanisms by which the viruses may overcome barriers in treating pediatric brain tumors, examine challenges associated with achieving effective, durable responses, highlight unique aspects and successes of the trials, and discuss future directions of immunovirotherapy research for the treatment of pediatric brain tumors.

Targeting oncometabolism to maximize immunotherapy in malignant brain tumors.

Brain tumors result in significant morbidity and mortality in both children and adults. Recent data indicate that immunotherapies may offer a survival benefit after standard of care has failed for malignant brain tumors. Modest results from several late phase clinical trials, however, underscore the need for more refined, comprehensive strategies that incorporate new mechanistic and pharmacologic knowledge. Recently, oncometabolism has emerged as an adjunct modality for combinatorial treatment approaches necessitated by the aggressive, refractory nature of high-grade glioma and other progressive malignant brain tumors. Manipulation of metabolic processes in cancer and immune cells that comprise the tumor microenvironment through controlled targeting of oncogenic pathways may be utilized to maximize the efficacy of immunotherapy and improve patient outcomes. Herein, we summarize preclinical and early phase clinical trial research of oncometabolism-based therapeutics that may augment immunotherapy by exploiting the biochemical and genetic underpinnings of brain tumors. We also examine metabolic pathways related to immune cells that target tumor cells, termed "tumor immunometabolism". Specifically, we focus on glycolysis and altered glucose metabolism, including glucose transporters, hexokinase, pyruvate dehydrogenase, and lactate dehydrogenase, glutamine, and we discuss targeting arginase, adenosine, and indoleamine 2,3-dioxygenase, and toll-like receptors. Lastly, we summarize future directions targeting metabolism in combination with emerging therapies such as oncolytic virotherapy, vaccines, and chimeric antigen receptor T cells.

Safety and Efficacy of Intraventricular Immunovirotherapy with Oncolytic HSV-1 for CNS Cancers.

PURPOSE: Oncolytic virotherapy with herpes simplex virus-1 (HSV) has shown promise for the treatment of pediatric and adult brain tumors; however, completed and ongoing clinical trials have utilized intratumoral/peritumoral oncolytic HSV (oHSV) inoculation due to intraventricular/intrathecal toxicity concerns. Intratumoral delivery requires an invasive neurosurgical procedure, limits repeat injections, and precludes direct targeting of metastatic and leptomeningeal disease. To address these limitations, we determined causes of toxicity from intraventricular oHSV and established methods for mitigating toxicity to treat disseminated brain tumors in mice. EXPERIMENTAL DESIGN: HSV-sensitive CBA/J mice received intraventricular vehicle, inactivated oHSV, or treatment doses (1×107 plaque-forming units) of oHSV, and toxicity was assessed by weight loss and IHC. Protective strategies to reduce oHSV toxicity, including intraventricular low-dose oHSV or interferon inducer polyinosinic-polycytidylic acid (poly I:C) prior to oHSV treatment dose, were evaluated and then utilized to assess intraventricular oHSV treatment of multiple models of disseminated CNS disease. RESULTS: A standard treatment dose of intraventricular oHSV damaged ependymal cells via virus replication and induction of CD8+ T cells, whereas vehicle or inactivated virus resulted in no toxicity. Subsequent doses of intraventricular oHSV caused little additional toxicity. Interferon induction with phosphorylation of eukaryotic initiation factor-2α (eIF2α) via intraventricular pretreatment with low-dose oHSV or poly I:C mitigated ependyma toxicity. This approach enabled the safe delivery of multiple treatment doses of clinically relevant oHSV G207 and prolonged survival in disseminated brain tumor models. CONCLUSIONS: Toxicity from intraventricular oHSV can be mitigated, resulting in therapeutic benefit. These data support the clinical translation of intraventricular G207.

Immunotherapy approaches for the treatment of diffuse midline gliomas.

Diffuse midline gliomas (DMG) are a highly aggressive and universally fatal subgroup of pediatric tumors responsible for the majority of childhood brain tumor deaths. Median overall survival is less than 12 months with a 90% mortality rate at 2 years from diagnosis. Research into the underlying tumor biology and numerous clinical trials have done little to change the invariably poor prognosis. Continued development of novel, efficacious therapeutic options for DMGs remains a critically important area of active investigation. Given that DMGs are not amenable to surgical resection, have only limited response to radiation, and are refractory to traditional chemotherapy, immunotherapy has emerged as a promising alternative treatment modality. This review summarizes the various immunotherapy-based treatments for DMG as well as their specific limitations. We explore the use of cell-based therapies, oncolytic virotherapy or immunovirotherapy, immune checkpoint inhibition, and immunomodulatory vaccination strategies, and highlight the recent clinical success of anti-GD2 CAR-T therapy in diffuse intrinsic pontine glioma (DIPG) patients. Finally, we address the challenges faced in translating preclinical and early phase clinical trial data into effective standardized treatment for DMG patients.

Effect of starvation upon baculovirus replication in larval Bombyx mori and Heliothis virescens.

The progression of baculovirus (BmNPV, BmCysPD, AcMNPV or AcAaIT) infection in larval Bombyx mori and Heliothis virescens (1st, 3rd or 5th instar) was investigated following various starvation regimes. When the larvae were starved for 12 or 24h immediately following inoculation, the median lethal time to death (LT(50)) was delayed by 9.5-19.2h in comparison to non-starved controls. This corresponded to a delay of 10-23% depending upon the larval stage and virus that was used for inoculation. When a 24h-long starvation period was initiated at 1 or 2 days post inoculation (p.i.), a statistically significant difference in LT(50) was not found indicating that the early stages of infection are more sensitive to the effects of starvation. Viral titers in the hemolymph of 5th instar B. mori that were starved for 24h immediately following inoculation were 10-fold lower (p<0.01) than that found in non-starved control larvae. Histochemical analyses indicated that virus transmission was reduced in 5th instar B. mori that were starved for 24h immediately following inoculation in comparison to non-starved control larvae. In general, the mass of larvae that were starved immediately after inoculation was 30% lower than that of non-starved control insects. Our findings indicate that starvation of the larval host at the time of baculovirus exposure has a negative effect on the rate baculovirus transmission and pathogenesis.

Oncolytic herpes simplex virus immunotherapy for brain tumors: current pitfalls and emerging strategies to overcome therapeutic resistance.

Malignant tumors of the central nervous system (CNS) continue to be a leading cause of cancer-related mortality in both children and adults. Traditional therapies for malignant brain tumors consist of surgical resection and adjuvant chemoradiation; such approaches are often associated with extreme morbidity. Accordingly, novel, targeted therapeutics for neoplasms of the CNS, such as immunotherapy with oncolytic engineered herpes simplex virus (HSV) therapy, are urgently warranted. Herein, we discuss treatment challenges related to HSV virotherapy delivery, entry, replication, and spread, and in so doing focus on host anti-viral immune responses and the immune microenvironment. Strategies to overcome such challenges including viral re-engineering, modulation of the immunoregulatory microenvironment and combinatorial therapies with virotherapy, such as checkpoint inhibitors, radiation, and vaccination, are also examined in detail.

Activation of the Receptor Tyrosine Kinase AXL Regulates the Immune Microenvironment in Glioblastoma.

Glioblastoma (GBM) is a lethal disease with no effective therapies available. We previously observed upregulation of the TAM (Tyro-3, Axl, and Mer) receptor tyrosine kinase family member AXL in mesenchymal GBM and showed that knockdown of AXL induced apoptosis of mesenchymal, but not proneural, glioma sphere cultures (GSC). In this study, we report that BGB324, a novel small molecule inhibitor of AXL, prolongs the survival of immunocompromised mice bearing GSC-derived mesenchymal GBM-like tumors. We show that protein S (PROS1), a known ligand of other TAM receptors, was secreted by tumor-associated macrophages/microglia and subsequently physically associated with and activated AXL in mesenchymal GSC. PROS1-driven phosphorylation of AXL (pAXL) induced NFκB activation in mesenchymal GSC, which was inhibited by BGB324 treatment. We also found that treatment of GSC-derived mouse GBM tumors with nivolumab, a blocking antibody against the immune checkpoint protein PD-1, increased intratumoral macrophages/microglia and activation of AXL. Combinatorial therapy with nivolumab plus BGB324 effectively prolonged the survival of mice bearing GBM tumors. Clinically, expression of AXL or PROS1 was associated with poor prognosis for patients with GBM. Our results suggest that the PROS1-AXL pathway regulates intrinsic mesenchymal signaling and the extrinsic immune microenvironment, contributing to the growth of aggressive GBM tumors.Significance: These findings suggest that development of combination treatments of AXL and immune checkpoint inhibitors may provide benefit to patients with GBM. Cancer Res; 78(11); 3002-13. ©2018 AACR.

Pyrethroid tolerance in Culex pipiens pipiens var molestus from Marin County, California.

In May 2001 a sample of Culex pipiens pipiens variety molestus Forskål from Marin County, California, collected as larvae and reared to adults, was found to show reduced resmethrin and permethrin knock-down responses in bottle bioassays relative to a standard susceptible Cx. pipiens quinquefasciatus Say colony (CQ1). Larval susceptibility tests, using CQ1 as standard susceptible, indicated that the Marin mosquitoes had LC50 resistance ratios of 18.3 for permethrin, 12 for deltamethrin and 3.3 for pyrethrum. A colony of Marin was established and rapidly developed higher levels of resistance in a few generations after exposure to permethrin as larvae. These selected larvae were shown to cross-resist to lambda-cyhalothrin as well as to DDT. However, adult knock-down time in the presence of permethrin, resmethrin and pyrethrum was not increased after increase in tolerance to pyrethroids as larvae. Partial and almost complete reversion to susceptibility as larvae was achieved with S, S, S-tributylphosphorotrithioate and piperonyl butoxide (PBO), respectively, suggesting the presence of carboxylesterase and P450 monooxygenase mediated resistance. Insensitive target site resistance (kdr) was also detected in some Marin mosquitoes by use of an existing PCR-based diagnostic assay designed for Cx. p. pipiens L mosquitoes. Carboxylesterase mediated resistance was supported by use of newly synthesized novel pyrethroid-selective substrates in activity assays. Bottle bioassays gave underestimates of the levels of tolerance to pyrethroids of Marin mosquitoes when compared with mortality rates in field trials using registered pyrethroid adulticides with and without PBO. This study represents the first report of resistance to pyrethroids in a feral population of a mosquito species in the USA.

Anti-hyperlipidemic effect of soybean extract fermented by Bacillus subtilis MORI in db/db mice.

The purpose of this study was to investigate the anti-hyperlipidemic effect of soy bean extract solution fermented by Bacillus subtilis MORI (BTD-1E) in obese db/db mice. Eight-week-old male db/db mice were administered 33.3 mg/kg BTD-1E solution orally once a day for four weeks. The BTD-1E group showed significantly lower body weight compared with the db control group (P<0.05). The BTD-1E group showed significantly lower serum total cholesterol and LDL cholesterol levels compared with the db control group, respectively (P<0.05, P<0.01). The BTD-1E group showed significantly decreased liver weight relative to final body weight compared with the db control group (P<0.01). After four weeks of BTD-1E administration, lipid droplets in the liver were apparently decreased in the BTD-1E group compared to the db control group. In summary, our results suggest that BTD-1E has an anti-hyperlipidemic effect in the obese mouse model.

Assessment of antidiabetogenic potential of fermented soybean extracts in streptozotocin-induced diabetic rat.

Most of the available drugs for the treatment of diabetes mellitus (DM) produce detrimental side effects, which has prompted an ongoing search for plant with the antidiabetic potential. The present study investigated the effect of soybean extracts fermented with Bacillus subtilis MORI, fermented soybean extracts (BTD-1) was investigated in streptozotocin (STZ)-induced diabetic rats. The possible effects of BTD-1 against hyperglycemia and free radical-mediated oxidative stress was investigated by assaying the plasma glucose level and the activity of enzymatic antioxidants, such as superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT), and malondialdehyde (MDA). A significant increase in the levels of both plasma glucose and reactive oxygen species (ROS) was observed in the diabetic rats when compared to normal control group. After administration of BTD-1 (500 and 1000 mg/kg/day), the elevated plasma glucose level was significantly reduced while the plasma insulin level and the activities of SOD, GSH-Px, CAT and MDA were significantly increased. The results suggest that administration of BTD-1 can inhibit hyperglycemia and free radical-mediated oxidative stress. The administration of BTD-1 also inhibited the contractile response by norepinephrine (10(-10)-10(-5) M) in the presence of endothelium, and caused significant relaxation by carbachol (10(-8)-10(-5) M) in rat aorta. These findings indicate that BTD-1 improves vascular functions on STZ-induced diabetic rats. Therefore, subchronic administration of BTD-1 could prevent the functional changes in vascular reactivity in STZ-induced diabetic rats. The collective findings support that administration of BTD-1 may prevent some diabetes-related changes in vascular reactivity directly and/or indirectly due to its hypoglycaemic effect and inhibition of production of ROS.

Identification of the genes involved in 1-deoxynojirimycin synthesis in Bacillus subtilis MORI 3K-85.

1-Deoxynojirimycin (DNJ), a D-glucose analogue with a nitrogen atom substituting for the ring oxygen, is a strong inhibitor of intestinal α-glucosidase. DNJ has several promising biological activities, including its antidiabetic, antitumor, and antiviral activities. Nevertheless, only limited amounts of DNJ are available because it can only be extracted from some higher plants, including the mulberry tree, or purified from the culture broth of several types of soil bacteria, such as Streptomyces sp. and Bacillus sp. In our previous study, a DNJ-producing bacterium, Bacillus subtilis MORI, was isolated from the traditional Korean fermented food Chungkookjang. In the present study, we report the identification of the DNJ biosynthetic genes in B. subtilis MORI 3K-85 strain, a DNJ-overproducing derivate of the B. subtilis MORI strain generated by γ-irradiation, xhe genomic DNA library of B. subtilis MORI 3K-85 was constructed in Escherichia coli, and clones showing α-glucosidase inhibition activity were selected. After DNA sequencing and a series of subcloning, we were able to identify a putative Operon which consists of gabT1, yktc1, and gutB1 genes predicted to encode putative transaminase, phosphatase, and oxidoreductase, respectively. When a recombinant plasmid containing this Operon sequence was transformed into an E. coli strain, the resulting transformant was able to produce DNJ into the culture medium. Our results indicate that the gabT1, yktc1, and gutB1 genes are involved in the DNJ biosynthetic pathway in B. subtilis MORI, suggesting the possibility of employing these genes to establish a large-scale microbial DNJ overproduction system through genetic engineering and process optimization.

Evaluation of alpha-cyano ethers as fluorescent substrates for assay of cytochrome P450 enzyme activity.

We have previously reported the synthesis of four alpha-cyano-containing ethers based on 2-naphthaldehyde (2-NA) as cytochrome P450 (P450) fluorescent substrates. Activity detection was based on the formation of fluorescent 2-NA following substrate hydrolysis. A major limitation of these substrates was the need to remove NADPH, a required cofactor for P450 oxidation, before measuring 2-NA fluorescence. In this article, we report the synthesis of a new series of novel P450 substrates using 6-dimethylamino-2-naphthaldehyde (6-DMANA), which has a green fluorescent emission that is well separated from the NADPH spectrum. A major advantage of the 6-DMANA substrates is that NADPH removal is not required before fluorescence detection. We used eight alpha-cyano ether-based substrates to determine the O-dealkylation activity of human, mouse, and rat liver microsomes. In addition, substrate activities were compared with the commercial substrate 7-ethoxyresorufin (7-ER). The catalytic turnover rates of both the 6-DMANA- and 2-NA-based substrates were in some cases threefold faster than the catalytic turnover rate of 7-ER. The 2-NA-based substrates had greater turnover than did the 6-DMANA-based substrates. Murine and rat liver microsomes prepared from animals that had been treated with various P450 inducers were used to examine for isozyme-selective turnover of the substrates. The vastly improved optical properties and synthetic flexibility of the alpha-cyano ether compounds suggest that they are possibly good general P450 substrates.