Fragment-Based Drug Discovery for RNA Targets.

Rapid development within the fields of both fragment-based drug discovery (FBDD) and medicinal targeting of RNA provides possibilities for combining technologies and methods in novel ways. This review provides an overview of fragment-based screening (FBS) against RNA targets, including a discussion of the most recently used screening and hit validation methods such as NMR spectroscopy, X-ray crystallography, and virtual screening methods. A discussion of fragment library design based on research from small-molecule RNA binders provides an overview on both the currently limited guidelines within RNA-targeting fragment library design, and future possibilities. Finally, future perspectives are provided on screening and hit validation methods not yet used in combination with both fragment screening and RNA targets.

Changes in melanocyte RNA and DNA methylation favour pheomelanin synthesis and may avoid systemic oxidative stress after dietary cysteine supplementation in birds.

Cysteine plays essential biological roles, but excessive amounts produce cellular oxidative stress. Cysteine metabolism is mainly mediated by the enzymes cysteine dioxygenase and γ-glutamylcysteine synthetase, respectively coded by the genes CDO1 and GCLC. Here we test a new hypothesis posing that the synthesis of the pigment pheomelanin also contributes to cysteine homeostasis in melanocytes, where cysteine can enter the pheomelanogenesis pathway. We conducted an experiment with the Eurasian nuthatch Sitta europaea, a bird producing large amounts of pheomelanin for feather pigmentation, to investigate if melanocytes show epigenetic lability under exposure to excess cysteine. We increased systemic cysteine levels in nuthatches by supplementing them with dietary cysteine during growth. In feather melanocytes this led to the downregulation of genes involved in intracellular cysteine metabolism (GCLC), cysteine transport to the cytosol from the extracellular medium (Slc7a11) and from melanosomes (CTNS), and regulation of tyrosinase activity (MC1R and ASIP). These changes were mediated by increases in DNA m5 C in all genes except Slc7a11, which experienced RNA m6 A depletion. Birds supplemented with cysteine synthesized more pheomelanin than controls, but did not suffer higher systemic oxidative stress. These results suggest that excess cysteine activates an epigenetic mechanism that favours pheomelanin synthesis and may protect against oxidative stress.

Prolotherapy Induces an Inflammatory Response in Human Tenocytes In Vitro.

BACKGROUND: Proliferative therapy, or prolotherapy, is a controversial treatment method for many connective tissue injuries and disorders. It involves the injection of a proliferant, or irritant solution, into the site of injury, which causes small-scale cell death. This therapeutic trauma is theorized to initiate the body's wound-healing cascade, perhaps leading to tissue repair. The immediate effects of many of these proliferants are poorly characterized, as are the cellular responses to them; here, we sought to evaluate the immediate effects of two common proliferants (dextrose and P2G, a combination of phenol, glucose, and glycerin) on the cellular response of human tenocytes, and begin to explicate the mechanisms with which each proliferant functions. QUESTIONS/PURPOSES: We asked: What are the effects of treating cultured tenocytes with proliferative treatment agents on their (1) cellular metabolic activity, (2) RNA expression, (3) protein secretion, and (4) cell migration? METHODS: Using human hamstring and Achilles tendon cells, we attempted to answer our research questions. We used a colorimetric metabolic assay to assess the effect of dextrose and P2G proliferant treatment on cell mitochondrial activity compared with nontreated tenocytes. Next, using quantitative PCR, ELISA, and a reporter cell line, we assessed the expression of several key markers involved in tendon development and inflammation. In addition, we used a scratch wound-healing assay to evaluate the effect of proliferant treatment on tenocyte migration. RESULTS: Results showed that exposure to both solutions led to decreased metabolic activity of tenocytes, with P2G having the more pronounced effect (75% ± 7% versus 95% ± 7% of untreated control cell metabolic levels) (ANOVA; p < 0.01; mean difference, 0.202; 95% CI, 0.052-0.35). Next, gene expression analysis confirmed that treatment led to the upregulation of key proinflammatory markers including interleukin-8 and cyclooxygenase-2 and downregulation of the matrix marker collagen type I. Furthermore, using a reporter cell line for transforming growth factor-ß (TGF-ß), a prominent antiinflammatory marker, we showed that treatments led to decreased TGF-ß bioactivity. Analysis of soluble proteins using ELISA revealed elevated levels of soluble prostaglandin E2 (PGE2), a prominent inducer of inflammation. Finally, both solutions led to decreased cellular migration in the tenocytes. CONCLUSIONS: Taken together, these results suggest that prolotherapy, more so with P2G, may work by decreasing cellular function and eliciting an inflammatory response in tenocytes. Additional studies are needed to confirm the cellular signaling mechanisms involved and the resulting immediate response in vivo. CLINICAL RELEVANCE: If these preliminary in vitro findings can be confirmed in an in vivo model, they may provide clues for a possible cellular mechanism of a common alternative treatment method currently used for certain soft tissue injuries.

Single-molecule study of the CUG repeat-MBNL1 interaction and its inhibition by small molecules.

Effective drug discovery and optimization can be accelerated by techniques capable of deconvoluting the complexities often present in targeted biological systems. We report a single-molecule approach to study the binding of an alternative splicing regulator, muscleblind-like 1 protein (MBNL1), to (CUG)n = 4,6 and the effect of small molecules on this interaction. Expanded CUG repeats (CUG(exp)) are the causative agent of myotonic dystrophy type 1 by sequestering MBNL1. MBNL1 is able to bind to the (CUG)n-inhibitor complex, indicating that the inhibition is not a straightforward competitive process. A simple ligand, highly selective for CUG(exp), was used to design a new dimeric ligand that binds to (CUG)n almost 50-fold more tightly and is more effective in destabilizing MBNL1-(CUG)4. The single-molecule method and the analysis framework might be extended to the study of other biomolecular interactions.

Immunostimulatory effects of gold nanorod and silica-coated gold nanorod on RAW 264.7 mouse macrophages.

In this study, we have investigated immunostimulatory effects of gold nanorod (Au) and silica-coated gold nanorod (SiAu) concerned with inflammatory mediators such as calcium (Ca), hydrogen peroxide, nitric oxide (NO), various cytokines, prostaglandin E2 (PGE2), transcription factors, and immune response gene in RAW 264.7 mouse macrophages. Both Au and SiAu significantly increased the release of Ca, hydrogen peroxide, NO, IL-1α, IL-1ß, IL-6, IL-10, IP-10, MCP-1, MCP-3, TNF-α, RANTES, G-CSF, GM-CSF, LIF, MIP-2, VEGF, and PGE2 with enhancing expression of STAT1, STAT3, c-Fos, and COX-2 mRNA in RAW 264.7 cells. The results suggest that Au and SiAu have immunostimulatory properties to strengthen immune reactions via calcium-transcription factors pathway in macrophage.

Regulation of fasting fuel metabolism by toll-like receptor 4.

OBJECTIVE: Toll-like receptor 4 (TLR4) has been reported to induce insulin resistance through inflammation in high-fat-fed mice. However, the physiological role of TLR4 in metabolism is unknown. Here, we investigated the involvement of TLR4 in fasting metabolism. RESEARCH DESIGN AND METHODS: Wild-type and TLR4 deficient (TLR4(-/-)) mice were either fed or fasted for 24 h. Glucose and lipid levels in circulation and tissues were measured. Glucose and lipid metabolism in tissues, as well as the expression of related enzymes, was examined. RESULTS: Mice lacking TLR4 displayed aggravated fasting hypoglycemia, along with normal hepatic gluconeogenesis, but reversed activity of pyruvate dehydrogenase complex (PDC) in skeletal muscle, which might account for the fasting hypoglycemia. TLR4(-/-) mice also exhibited higher lipid levels in circulation and skeletal muscle after fasting and reversed expression of lipogenic enzymes in skeletal muscle but not liver and adipose tissue. Adipose tissue lipolysis is normal and muscle fatty acid oxidation is increased in TLR4(-/-) mice after fasting. Inhibition of fatty acid synthesis in TLR4(-/-) mice abolished hyperlipidemia, hypoglycemia, and PDC activity increase, suggesting that TLR4-dependent inhibition of muscle lipogenesis may contribute to glucose and lipid homeostasis during fasting. Further studies showed that TLR4 deficiency had no effect on insulin signaling and muscle proinflammatory cytokine production in response to fasting. CONCLUSIONS: These data suggest that TLR4 plays a critical role in glucose and lipid metabolism independent of insulin during fasting and identify a novel physiological role for TLR4 in fuel homeostasis.

Vitamin a supplements alleviate inflammatory responses in reproductive tracts of male mice infected with pseudorabies virus.

Vitamin A is largely thought to have immune potential for mammal health; however, no conclusive mechanisms exist regarding its role in the pathogen-initiated innate immune response, or in the linkage between the innate and adaptive immune system during sperm formation in the male reproductive tract. Therefore, this study was conducted to evaluate the nutritional protective effect of vitamin A supplementation on reproductive performance and immune function of the male mouse challenged with pseudorabies virus (PRV). Sperm quality, testis toll-like receptors (TLRs) mRNA expression levels, and serum concentration of cytokines and immunoglobulins at 7 or 14 days post-injection were compared between control mice and PRV-challenged mice fed the same diet supplemented with vitamin A at 0, 4000, 10,000, 25,000 and 50,000 IU/kg, respectively. PRV- and phosphate buffered saline (PBS)-injection were performed when the mice in the unsupplemented group were marginally deficient in vitamin A. Sperm quality (sperm density and deformity ratio) of PRV-injected mice was significantly harmed by PRV, but this effect was attenuated by increased vitamin A consumption. Vitamin A supplements also attenuated PRV-challenge-induced increase in testis TLR3, TLR7, and TLR9 mRNA expression and serum pro-inflammatory cytokine (gamma interferon, IFN-gamma; and interleukin 1-beta,IL-1beta) concentration, and decrease in serum anti-inflammatory cytokine (IL-10) concentration. Higher than normal vitamin A consumption was recommended to counteract the deleterious effects of viral invasion, possibly through the downregulated expression of TLRs, and thus to improve immunity and reproductivity of male mice challenged with an invading pathogen.

Toxic effects of crotocaudin extracted from the medicinal plant Croton tiglium.

The compound crotocaudin extracted from the stem bark of the medicinal plant Croton tiglium Linn. was administered for 24 h or 96 h to the freshwater vector snail Lymnaea (Radix) acuminata Lamarck in order to test its toxicity. L. acuminata is the intermediate host of Fasciola hepatica and Fasciola gigantica which cause immense harm to man and his domestic animals. It was observed that the molluscicidal activity of crotocaudin against L. acuminata is time- as well as dose-dependent. There was a significant negative correlation among LC50 values and exposure periods, i.e. increasing the exposure time, the LC50 value of crotocaudin decreased from 5.37 microM (24 h) > 2.08 microM (48 h) > 1.36 microM (72 h) to 1.01 microM (96 h), respectively, against L. acuminata. The toxicological experiments to proof for environmental toxicity, if any, have also been carried out on the non-target freshwater fish Channa punctatus (Bloch) [Channidae (Ophicephalidae)], which shares the habitat with L. acuminata. The sublethal doses of crotocaudin (40% and 80% of LC50) administered over 24 h caused significant changes in the carbohydrate and nitrogenous metabolisms in nervous, hepatopancreas, and ovotestis tissues of Lymnaea acuminata. Channa punctatus was also exposed to sublethal doses of crotocaudin (40% and 80% of 24-h LC50 of L. acuminata) for 96 h which showed significant alterations in the metabolism in muscle, liver, and gonad tissues. After withdrawal of crotocaudin the snail tissues recovered in part after 7 days and the fish tissues completely.

Endoplasmic reticulum stress induced by tunicamycin and antagonistic effect of Tiantai No.1 (1) on mesenchymal stem cells.

OBJECTIVE: Changes of the internal and external cellular environments can induce calcium homeostasis disorder and unfolded protein aggregation in the endoplasmic reticulum (ER). This ER function disorder is called endoplasmic reticulum stress (ERS). Severe long-term ERS can trigger the ER apoptosis signaling pathway, resulting in cell apoptosis and organism injury. Recent researches revealed that ERS-induced cell death was involved in the neurocyte retrogradation in the progress of neuron degenerative diseases, such as Alzheimer's disease (AD), Parkinson's disease and so on. Therefore, the protection effect of the traditional Chinese drug-Tiantai No. 1 (1) on the ERS injury of AD was investigated at the molecular gene level in this study with a view to explore the gene pharmacodynamic actions and mechanisms of this drug. METHODS: Primarily cultured marrow mesenchymal stem cells (MSCs) of rats were treated by tunicamycin (TM) in order to induce ERS. RT-PCR, fluorescence immunocytochemistry and Western blot techniques were used to determine the mRNA and protein expression levels of the protective stress protein-ER molecular chaperones GRP78 and GRP94 (which would assist cells to resist cellular stress injury), and to determine the mRNA and protein expression levels of apoptosis promoting molecule Caspase-12 on the membrane of the ER, respectively. RESULTS: Protein expression levels of GRP78 and GRP94 were significantly increased in the TM-induced MSCs, and the mRNA level of Caspase-12 was also remarkably increased in the TM-induced MSCs (P<0.05). All these proved that the ERS model was successfully established by TM in MSC. Meanwhile, the mRNA and protein levels of GRP78 and GRP94 were all significantly increased compared with the model group (P<0.05 or P<0.01) after MSCs were treated with Tiantai No.1 while the mRNA and protein expression levels of Caspase-12 were significantly decreased compared with the model group (P<0.05 or P<0.01). This effect showed a dose dependent manner. CONCLUSION: Tiantai No.1 might attenuate the cell apoptosis induced by ERS injury, and thus protect the neurons against AD.

RNA as a new target for toxic and protective agents.

In contrast to damage of genomic DNA and despite its potential to affect cell physiology, RNA damage is a poorly examined field in biomedical research. Potential triggers of RNA damage as well as its pathophysiological implications remain largely unknown. While less lethal than mutations in genome, such non-acutely lethal insults to cells have been recently associated with underlying mechanisms of several human chronic diseases. We investigated whether RNA damage could be related to the exposure of particular xenobiotics by testing the RNA-damaging activity of a series of chemicals with different mechanisms of action. Cultured human T-lymphoblastoid cells were treated with ethyl methanesulfonate (EMS), H(2)O(2), doxorubicin, spermine, or S-nitroso-N-acetylpenicillamine (SNAP). Furthermore, we studied the potential protective activity of a pomegranate extract against RNA damage induced by different chemicals. Special attention has been paid to the protective mechanisms of the extract. The protective effect of pomegranate can be mediated by alterations of the rates of toxic agent absorption and uptake, by trapping of electrophiles as well as free radicals, and protection of nucleophilic sites in RNA. We used two different treatment protocols (pre- and co-treatment) for understanding the mechanism of the inhibitory activity of pomegranate. We demonstrated that total RNA is susceptible to chemical attack. A degradation of total RNA could be accomplished with doxorubicin, H(2)O(2), spermine and SNAP. However, EMS, a well-known DNA-damaging agent, was devoid of RNA-damaging properties, while spermine and SNAP, although lacking of DNA-damaging properties, were able to damage RNA. Pomegranate reduced the RNA-damaging effect of doxorubicin, H(2)O(2), and spermine. Its inhibitory activity could be related with its ability to forms complexes with doxorubicin and H(2)O(2), or interacts with the intracellular formation of reactive species mediating their toxicity. For spermine, an alteration of the rates of spermine absorption and uptake can also be involved.

Eicosapentaenoic acid inhibits interleukin-6 production in interleukin-1beta-stimulated C6 glioma cells through peroxisome proliferator-activated receptor-gamma.

Epidemiological studies suggest that intake of omega-3 polyunsaturated fatty acids improves neurological disorders such as Alzheimer's disease which exhibit inflammatory pathology. We therefore investigated the anti-inflammatory effects of eicosapentaenoic acid (EPA) on interleukin (IL)-1beta-stimulated C6 glioma cells. In the present study, EPA inhibited pro-inflammatory cytokine IL-6 production, a characteristic of certain neurodegenerative disorders, in IL-1beta-stimulated C6 glioma cells in dose-dependent fashion. EPA down-regulated the expression of IL-6 at mRNA level, indicating that the effect of EPA occurs at the transcriptional level. In addition, peroxisome proliferator-activated receptor (PPAR) gamma antagonists abolished the inhibitory effect of EPA on IL-1beta-induced IL-6 production, whereas PPARalpha antagonist did not block the inhibitory effect of EPA. EPA might thus contribute to the regulation of pro-inflammatory cytokine production in astrocytes through interaction with PPARgamma. Among the PPARgamma ligands tested in this study, ciglitazone, a synthetic agonist of PPARgamma, effectively inhibited IL-6 production, but while neither rosiglitazone nor 15-deoxy-Delta(12,14)-prostaglandin J2 did. These findings indicate that the coordination of PPAR gamma ligands is important in inhibiting the production of IL-6 in C6 glioma cells.

Alterations in DNA, RNA and protein contents in liver and kidney of mice treated with ochratoxin and their amelioration by Emblica officinalis aqueous extract.

The present investigation was an attempt to study ameliorative effect of Emblica officinalis aqueous extract on ochratoxin-induced toxicity on DNA, RNA and protein contents in liver and kidney of mice. Adult male albino mice were orally administered with 50 and 100 microg of ochratoxin/0.2 mL of olive oil/animal/day for 45 days. The results revealed significant (p < 0.05), dose-dependent decrease in the concentrations of DNA, RNA as well as total, acidic, basic and neutral protein contents in the liver and kidney of ochratoxin-treated mice. Administration of Emblica officinalis aqueous extract (2 mg/animal/day) for 45 days along with ochratoxin caused significant amelioration in the ochratoxin-induced reduction in DNA, RNA and protein contents in the liver and kidney of mice.

Ameliorative effect of curcumin on aflatoxin-induced toxicity in DNA, RNA and protein in liver and kidney of mice.

The present investigation is an attempt to evaluate the ameliorative effect of curcumin on aflatoxin-induced toxicity in liver and kidney of mice. Aflatoxin was obtained by growing Aspergillus parasiticus in SMKY liquid medium. 70 male mice were divided into 7 groups (37-40 g body weight) including untreated control, vehicle control (0.2 mL olive oil/animal/day), curcumin control (50 mg/kg body weight/animal), aflatoxin low dose and high dose (750 and 1500 mg/kg body weight). Other two groups were administered curcumin along with low dose aflatoxin and high dose aflatoxin. The treatment was given for 45 days. On 46th day the animals were sacrificed by cervical dislocation. Liver and kidney were removed and weighed. Homogenates were prepared and analyzed for DNA, RNA and protein content. The results revealed dose-dependent significant reduction in DNA, RNA and protein contents in the liver and kidney of mice. Oral administration of aflatoxin along with curcumin significantly ameliorates, as compared to aflatoxin alone treated groups, in all parameters. It is concluded that curcumin ameliorates aflatoxin-induced toxicity in liver and kidney of mice.

Effect of olive oils on biomarkers of oxidative DNA stress in Northern and Southern Europeans.

High consumption of olive oil in the Mediterranean diet has been suggested to protect DNA against oxidative damage and to reduce cancer incidence. We investigated the impact of the phenolic compounds in olive oil, and the oil proper, on DNA and RNA oxidation in North, Central, and South European populations. In a multicenter, double-blind, randomized, controlled crossover intervention trial, the effect of olive oil phenolic content on urinary oxidation products of guanine (8-oxo-guanine, 8-oxo-guanosine and 8-oxo-deoxyguanosine) was investigated. Twenty-five milliliters of three olive oils with low, medium, and high phenolic content were administered to healthy males (n=182) daily for 3 wk. At study baseline the urinary excretion of 8-oxo-guanosine (RNA oxidation) and 8-oxo-deoxyguanosine (DNA oxidation) was higher in the Northern regions of Europe compared with Central and Southern European regions (P=0.035). Urinary excretion of the 8 hydroxylated forms of guanine, guanosine, deoxyguanosine and their nonoxidized forms were not different when comparing olive oils with low, medium, and high phenolic content given for 2 wk. Testing the effect of oil from urinary 8-oxo-deoxyguanosine changes from baseline to post-treatment showed a reduction of DNA oxidation by 13% (P=0.008). These findings support the idea that ingestion of olive oil is beneficial and can reduce the rate of oxidation of DNA. This effect is not due to the phenolic content in the olive oil. The higher DNA and RNA oxidation in Northern European regions compared with that in Central and Southern regions supports the contention that olive oil consumption may explain some of the North-South differences in cancer incidences in Europe.

Assessment of the DNA damaging potency and chemopreventive effects towards BaP-induced genotoxicity in human derived cells by Monimiastrum globosum, an endemic Mauritian plant.

Naturally occurring compounds have protective effects towards mutagens and carcinogens. The leaf extract of Monimiastrum globosum (Bois de Clous), a Mauritian endemic plant from the Myrtaceae family, was studied for its potency to induce DNA damage in human HepG2 hepatoma cells using DNA migration as a biological endpoint in the alkaline single cell gel electrophoresis (SCGE) assay. This was contrasted with the ability to modulate the benzo[a]pyrene (BaP)-dependent DNA damage in human hepatoma cells. M. globosum caused genotoxicity in HepG2 cells at concentrations exceeding 3mg fresh weight (FW) per ml cell culture in the absence of cytotoxicity. Pre-treatment of the cells with 12.2 microg FW/ml to 1.56 mg FW/ml led to a pronounced antigenotoxic effect towards BaP-induced DNA damage. DNA migration (OTM) was reduced by 66%, 81.5% and 74% for 49, 98 and 195 microg FW/ml, respectively. A U-shaped dose-response curve was derived for M. globosum indicating genotoxic effects in high doses and antigenotoxic effects in low doses. M. globosum extract had total phenolics (15 mg/g FW) with flavonoids (aglycones and conjugates: 8 mg/g FW) and proanthocyanidins (3mg/g FW) as major phenolic subclasses. The hydrolysis of conjugated flavonoids yielded the aglycones quercetin (606 microg/g FW) and kaempferol (117.8 microg/g FW) while HPLC-MS/MS analysis of the total extract revealed free flavonoids such as quercetin (19.2 microg/g FW) and myricetin (2.5 microg/g FW). The antioxidant activity of the extract of M. globosum, assessed by the FRAP and TEAC assays yielded values of 275+/-3.82 micromol/g FW and 346+/-4.2 micromol/g FW, respectively.

Effects of genistein on the mammary gland proliferation of adult ovariectomised Wistar rats.

The effects of phytoestrogens on the female breast are discussed controversially. On the one hand, epidemiological and experimental data provide evidence that dietary phytoestrogens may prevent the development of breast cancer. On the other hand, in breast cancer cell lines and tumour models isoflavone phytoestrogens have been demonstrated to stimulate the growth of estrogen-dependent breast cancer cells. To further investigate the molecular effects of genistein (Gen) on the mammary gland, we treated non-tumour bearing, ovariectomised female Wistar rats with this phytoestrogen either subcutaneously (10 mg/kg body weight) or orally (100 and 200 mg/kg body weight) for 3 days. Estradiol (E(2), 0.004 mg/kg s. c.) and ethynylestradiol (EE, 0.1 mg/kg per os) served as reference compounds. In the breast tissue, mRNA and protein expression of the progesterone receptor (marker for estrogenicity) and PCNA (marker gene for proliferation) were examined by quantitative real-time PCR, Western blotting and immunohistochemistry; the uterotrophic response was assessed also. Treatment with Gen per os or s. c. results in a small but significant stimulation of the uterine wet weight. In the mammary gland, Gen stimulates the expression of progesterone receptor (PR) but, in contrast to E(2), the isoflavone does not stimulate the expression of PCNA. These findings resemble recent data demonstrating a differential ability of Gen to induce uterine gene expression and uterine proliferation. Our data indicate that in non-malignant breast tissue short-term administration of Gen, in contrast to more potent estrogens like E(2), does not induce proliferation. Chronic stimulation of proliferation is believed to be a key mechanism during the development of breast cancer. The limited ability of Gen to stimulate proliferation in this tissue could be an indication for a limited carcinogenic potency of Gen in the breast. In further investigations it is important to identify molecular differences between healthy and malignant breast tissue which may explain the different sensitivity towards Gen treatment.

Radiomodulatory effect of liposome encapsulated AK-2123 on tumor in mice exposed to hepatocarcinogen.

An attempt was made to evaluate the whole body gamma-radiation effect on tumor in the presence of free and liposome encapsulated AK-2123, a hypoxic cell radiosensitizer that has widely been used in combination with a number of cancer therapies such as thermotherapy, chemotherapy and radiotherapy. Entrapment efficiency of AK-2123 into liposome was determined by LASER Raman spectroscopy. Cancer induction in mice was carried out by repeated exposure of N-nitrosodiethylamine (DEN) in combination with partial hepatectomy. Parameters such as marker enzymes activities (GGT and AChE), rates of nucleic acid synthesis, viability modification factor and the histology of liver tissues monitored, supported the induction of cancer in liver. In addition, the effect of free as well as liposome encapsulated AK-2123 on haemopoietic parameters were also studied. It was observed that AK-2123 after incorporation into liposome afforded more efficient radiomodulatory effects than that of free AK-2123 as determined by the above-mentioned parameters. Neither free AK-2123 nor liposome encapsulated AK-2123 showed any detectable toxic effects on the mice. Thus, it is seen that treatment of cancer with a combination of radiation, a radiomodifier and a drug delivery system, opens a wide scope for exploitation for the improvement of existing cancer therapies.

1,4,8-trimethylfuro[2,3-H]quinolin-2(1H)-one, a new furocoumarin bioisoster.

1,4,8-Trimethylfuro[2,3-h]quinolin-2(1H)-one (compound 5a) is the most interesting derivative among some new furoquinolinones prepared with the aim of moderating the strong toxic effects of 1,4,6,8-tetramethyl derivative (FQ), a powerful potential drug for photomedicine. Compound 5a showed a photobiological activity lower than FQ, but considerable higher than 8-MOP, the furocoumarin used in clinical photomedicine; contrary to classic furocoumarins, 5a induced a strong inhibition of protein synthesis in mammalian cells. Genotoxicity and skin erythema induction, the main side effects of both FQ and 8-MOP photosensitization, are virtually absent with 5a. This behavior seems to be connected to its particular reaction mechanism: differently from furocoumarin derivatives, 5a induced low levels of DNA-protein and no inter-strands cross-links, but formed covalent RNA-protein linkages, lesions not observed with known furocoumarins. Moreover, compound 5a generated reactive oxygen species to a considerable extent. For these features, compound 5a appears to be a new photosensitizing agent whose special activity deserves to be deeply investigated.

Suppressive effect of viscous dietary fiber on elevations of uric acid in serum and urine induced by dietary RNA in rats is associated with strength of viscosity.

This study was performed to clarify how dietary fiber (DF) with different viscosities would be associated with dietary RNA metabolism. Male Wistar strain rats, four weeks old, were fed diets containing a 3% (w/w) yeast RNA and a 5% (w/w) viscous DF for five days. Viscosity of DF samples used, in order of strength, were xanthan gum (XG) > guar gum (GG) > locust bean gum (LBG) > karaya gum (KG) > pectin (PE) = arabic gum (AG) > CM-cellulose (CMC) = inulin (IN). The serum uric acid concentration in the viscous DF groups significantly decreased as compared with that in the cellulose (CL) group. The urinary excretions of uric acid and allantoin in the respective groups given AG, GG, IN, KG, PE, and XG were significantly suppressed as compared with those in the CL group. The fecal RNA excretion was markedly increased in the IN, KG, PE, and XG groups in comparison to the CL group. The DF with high viscosity significantly suppressed RNA digestion by RNase A and decreased uptakes of 14C-labeled adenosine and adenosine 5'-monophosphate (5'-AMP) in rat jejunum. The results reveal that the suppressive effect of DF on elevation of serum uric acid concentration induced by dietary RNA in rats is associated with the strength of DF viscosity. The mechanism by which this is accomplished is suggested to be attributed to the inhibitions of digestion for dietary RNA and/or absorption of the hydrolyzed compounds.

Kissing complex-mediated dimerisation of HIV-1 RNA: coupling extended duplex formation to ribozyme cleavage.

Initiation of retroviral genomic RNA dimerisation is mediated by the mutual interaction of the dimerisation initiation site (DIS) stem-loops near to the 5' end of the RNA. This process is thought to involve formation of a transient 'kissing' complex over the self-complementary loop bases, which then refolds into a more stable extended interaction. We have developed a novel experimental system that allows us to clearly detect the extended duplex in vitro. Ribozyme sequences were incorporated into or adjacent to the type 1 human immunodeficiency virus DIS stem, leading to the formation of a functional ribozyme only in the extended duplex conformer. Here we show that extended duplex formation results in ribozyme cleavage, thus demonstrating the double-stranded nature of the extended complex and confirming that refolding occurs via melting of the DIS stems. Loop complementarity is essential for extended duplex formation but no sequence requirements for the loops were observed. Efficiency of extended duplex formation is dependent on the strength of the loop-loop interaction, temperature, the magnesium concentration and is strongly accelerated by the viral nucleocapsid protein NCp7. Our ribozyme-coupled approach should be applicable to the analyses of other refolding processes involving RNA loop-loop interactions.