Impact of dengue virus (serotype DENV-2) infection on liver of BALB/c mice: A histopathological analysis

In this research, we characterized the histopathological impact of dengue virus (serotype DENV-2) infection in livers of BALB/c mice. The mice were infected with different doses of DENV-2 via intraperitoneal injection and liver tissues were processed for histological analyses and variation was documented. In the BALB/c mouse model, typical liver tissues showed regular hepatocyte architecture, with normal endothelial cells surrounding sinusoid capillary. Based on histopathological observations, the liver sections of BALB/c mice infected by DENV-2 exhibited a loss of cell integrity, with a widening of the sinusoidal spaces. There were marked increases in the infiltration of mononuclear cells. The areas of hemorrhage and micro- and macrovesicular steatosis were noted. Necrosis and apoptosis were abundantly present. The hallmark of viral infection, i.e., cytopathic effects, included intracellular edema and vacuole formation, cumulatively led to sinusoidal and lobular collapse in the liver. The histopathological studies on autopsy specimens of fatal human DENV cases are important to shed light on tissue damage for preventive and treatment modalities, in order to manage future DENV infections. In this framework, the method present here on BALB/c mouse model may be used to study not only the effects of infections by other DENV serotypes, but also to investigate the effects of novel drugs, such as recently developed nano-formulations, and the relative recovery ability with intact immune functions of host.     

Acridones circumvent P-glycoprotein-associated multidrug resistance (MDR) in cancer cells

Multidrug resistance (MDR) mediated by overexpression of MDR1 P-glycoprotein (P-gp) is one of the best characterized transporter-mediated barriers to successful chemotherapy in cancer patients. Chemosensitizers are the agents that increase the sensitivity of multidrug-resistant cells to the toxic influence of previously less effective drugs. In an attempt to find such vital chemosensitizers, a series of N(10)-substituted-2-chloroacridone analogous (1-17) have been synthesized. Compound 1 was prepared by the Ullmann condensation of o-chlorobenzoic acid and p-chloroaniline followed by cyclization. The N-(omega-chloroalkyl) analogues were found to undergo iodide catalyzed nucleophilic substitution reaction with secondary amines and the resultant products were characterized by spectral methods. The lipophilicity expressed in log(10)P and pK(a) of compounds has been determined. All compounds were examined for their ability to increase the uptake of vinblastine (VLB) in MDR KBCh(R)-8-5 cells and the results showed that the compounds 6, 8, 11-14, 16, and 17 at their respective IC(50) concentrations caused a 1.0- to 1.7-fold greater accumulation of VLB than did a similar concentration of the standard modulator, verapamil (VRP). Results of the efflux experiment showed that VRP and each of the modulators significantly inhibited the efflux of VLB, suggesting that they may be competitors for P-gp. All modulators effectively competing with [(3)H]azidopine for binding to P-gp pointed out this transport membrane protein as their likely site of action. Compounds at IC(10) were evaluated for their efficacy to modulate the cytotoxicity of VLB and the results showed that modulators 11, 13, 14, 16, and 17 were able to completely reverse the 25-fold resistance of KBCh(R)-8-5 cells to VLB. Examination of the relationship between lipophilicity and antagonism of MDR showed a reasonable correlation suggesting that hydrophobicity is one of the determinants of potency for anti-MDR activity of 2-chloroacridones. The results allowed us to draw preliminary conclusions about structural features of 2-chloroacridones important for MDR modulation.     

Cell Proliferation and Epidermal Growth Factor Signaling in Non-small Cell Lung Adenocarcinoma Cell Lines Are Dependent on Rin1

Rin1 is a Rab5 guanine nucleotide exchange factor that plays an important role in Ras-activated endocytosis and growth factor receptor trafficking in fibroblasts. In this study, we show that Rin1 is expressed at high levels in a large number of non-small cell lung adenocarcinoma cell lines, including Hop62, H650, HCC4006, HCC827, EKVX, HCC2935, and A549. Rin1 depletion from A549 cells resulted in a decrease in cell proliferation that was correlated to a decrease in epidermal growth factor receptor (EGFR) signaling. Expression of wild type Rin1 but not the Rab5 guanine nucleotide exchange factor-deficient Rin1 (Rin1Δ) complemented the Rin1 depletion effects, and overexpression of Rin1Δ had a dominant negative effect on cell proliferation. Rin1 depletion stabilized the cell surface levels of EGFR, suggesting that internalization was necessary for robust signaling in A549 cells. In support of this conclusion, introduction of either dominant negative Rab5 or dominant negative dynamin decreased A549 proliferation and EGFR signaling. These data demonstrate that proper internalization and endocytic trafficking are critical for EGFR-mediated signaling in A549 cells and suggest that up-regulation of Rin1 in A549 cell lines may contribute to their proliferative nature.Internalization of epidermal growth factor receptors (EGFR)² and their subsequent delivery to lysosomes play key roles in attenuating EGF-mediated signaling cascades (1, 2). The proper delivery of EGFR into lysosomes for degradation requires a series of highly regulated targeting and delivery events. Following ligand binding, EGFR is internalized via endocytic vesicles that are subsequently targeted to early endosomes. This targeting event is mediated by the small GTPase, Rab5 (3, 4). Once delivered to the early endosome, receptors that are destined for degradation are incorporated into vesicles that bud into the lumen of the endosome, forming the multivesicular body (reviewed in Refs. 5, 6). Sequestration of the activated cytoplasmic domain of EGFR into the intralumenal vesicles of the multivesicular body effectively terminates receptor signaling (7). Subsequent fusion of the multivesicular body with lysosomes delivers the intralumenal vesicles and their contents into the lumen of the lysosome where they are degraded (reviewed in Refs. 8–10). Inactivating mutations in Rab5 disrupt the delivery of cell surface receptors, such as EGFR, to early endosomes, thereby inhibiting receptor trafficking to the lysosome and receptor degradation (11, 12). Therefore, activation of Rab5 is a key point of regulation for EGFR signaling.Rab5 cycles between an inactive GDP-bound state and an active GTP-bound state, and Rab5 activation requires the exchange of GDP to GTP. This exchange is catalyzed by guanine nucleotide exchange factors (GEFs) that are specific to the Rab5 family of proteins (reviewed in Ref. 13). Rab5 family GEFs all contain a catalytic vacuolar protein sorting 9 (Vps9) domain that facilitates the GDP to GTP exchange (14–17). Many Rab5 GEFs contain other functional domains that are involved in cell signaling events (13). Rin1 is a good example of a multidomain Rab5 GEF. In addition to the Vps9 domain, Rin1 also contains an Src homology 2 domain, a proline-rich domain, and a Ras association domain. Rin1 was originally identified through its ability to interact with active Ras (18), and a role for Rin1 in a number of cell signaling systems has been established, including EGF-mediated signaling (19–21). Rin1 directly interacts with the activated EGFR through its Src homology 2 domain (22). Furthermore, Ras occupation of the Rin1 Ras association domain positively impacts the Rab5 GEF activity of Rin1, which promotes EGFR internalization and attenuation in fibroblasts (23). However, Rin1 expression is up-regulated in several types of cancers, including squamous cell carcinoma (24), colorectal cancer (25), and cervical cancer (26), through duplications or rearrangements of the RIN1 locus. These studies suggest that Rin1 may also play a role in enhancing cell proliferation.It is well established that a large percentage of non-small cell lung adenocarcinomas exhibit up-regulation of EGFR and aberrant signaling through the Ras/MAPK pathway (reviewed in Ref. 27). In addition, a recent study examining 188 human lung adenocarcinomas identified that 132 of 188 tumor samples exhibited mutations relating to the Ras/MAPK signaling pathway (28). Accordingly, the role of Rin1 in non-small cell lung adenocarcinoma was addressed. Examination of a panel of non-small cell lung adenocarcinoma lines (including A549) revealed enhanced Rin1 expression relative to a nontransformed lung epithelial cell line (BEAS-2B). Depletion of Rin1 from A549 cells resulted in decreased proliferation. This decrease correlated with a reduction in EGF-activated ERK phosphorylation and the stabilization of cell surface EGFR. These defects were complemented by wild type Rin1 expression but not by mutant Rin1 lacking a functional Vps9 domain, suggesting that the GEF activity of Rin1 is necessary for proper EGFR signaling in A549 cells. In addition, overexpression of Rin1Δ, dominant negative Rab5, and dynamin resulted in similar defects in cell proliferation and EGFR signaling as Rin1 depletion. These data indicate that proper EGFR internalization and trafficking are critical for robust EGFR-mediated signaling and cell proliferation in A549 cells and offer evidence that Rin1 positively regulates cell proliferation in non-small cell lung adenocarcinoma.     

The biological activity and metabolic stability of peptidic bifunctional compounds that are opioid receptor agonists and neurokinin-1 receptor antagonists with a cystine moiety

In order to improve metabolic stability, a ring structure with a cystine moiety was introduced into TY027 (Tyr-d-Ala-Gly-Phe-Met-Pro-Leu-Trp-NH-[3′,5′-(CF₃)₂Bzl]), which is a lead compound of our developing bifunctional peptide possessing opioid agonist and NK1 antagonist activities. TY038 (Tyr-cyclo[d-Cys-Gly-Phe-Met-Pro-d-Cys]-Trp-NH-[3′,5′-(CF₃)₂Bzl]) was found as a highly selective δ opioid agonist over μ receptor in conventional tissue-based assays, together with an effective NK1 antagonist activity and good metabolic stability with more than 24 h half life in rat plasma.     

C19-steroids as androgen receptor modulators: design, discovery, and structure-activity relationship of new steroidal androgen receptor antagonists

Dehydroepiandrosterone (DHEA), the most abundant steroid in human circulating blood, is metabolized to sex hormones and other C19-steroids. Our previous collaborative study demonstrated that androst-5-ene-3beta,17beta-diol (Adiol) and androst-4-ene-3,17-dione (Adione), metabolites of DHEA, can activate androgen receptor (AR) target genes. Adiol is maintained at a high concentration in prostate cancer tissue; even after androgen deprivation therapy and its androgen activity is not inhibited by the antiandrogens currently used to treat prostate cancer patients. We have synthesized possible metabolites of DHEA and several synthetic analogues and evaluated their role in androgen receptor transactivation to identify AR modulators. Steroids with low androgenic potential in PC-3 cell lines were evaluated for anti-dihydrotestosterone (DHT) and anti-Adiol activity. We discovered three potent antiandrogens: 3beta-acetoxyandrosta-1,5-diene-17-one 17-ethylene ketal (ADEK), androsta-1,4-diene-3,17-dione 17-ethylene ketal (OAK), and 3beta-hydroxyandrosta-5,16-diene (HAD) that antagonized the effects of DHT as well as of Adiol on the growth of LNCaP cells and on the expression of prostate-specific antigen (PSA). In vivo tests of these compounds will reveal their potential as potent antiandrogens for the treatment of prostate cancer.     

T Cell receptor clonotype influences epitope hierarchy in the CD8+ T cell response to respiratory syncytial virus infection

CD8+ T cell responses are important for recognizing and resolving viral infections. To better understand the selection and hierarchy of virus-specific T cell responses, we compared the T cell receptor (TCR) clonotype in parent and hybrid strains of respiratory syncytial virus-infected mice. K(d)M2(82-90) (SYIGSINNI) in BALB/c and D(b)M(187-195) (NAITNAKII) in C57Bl/6 are both dominant epitopes in parent strains but assume a distinct hierarchy, with K(d)M2(82-90) dominant to D(b)M(187-195) in hybrid CB6F1/J mice. The dominant K(d)M2(82-90) response is relatively public and is restricted primarily to the highly prevalent Vβ13.2 in BALB/c and hybrid mice, whereas D(b)M(187-195) responses in C57BL/6 mice are relatively private and involve multiple Vβ subtypes, some of which are lost in hybrids. A significant frequency of TCR CDR3 sequences in the D(b)M(187-195) response have a distinct "(D/E)WG" motif formed by a limited number of recombination strategies. Modeling of the dominant epitope suggested a flat, featureless structure, but D(b)M(187-195) showed a distinctive structure formed by Lys(7). The data suggest that common recombination events in prevalent Vβ genes may provide a numerical advantage in the T cell response and that distinct epitope structures may impose more limited options for successful TCR selection. Defining how epitope structure is interpreted to inform T cell function will improve the design of future gene-based vaccines.     

Synthesis and chemical characterization of 2-methoxy-N(10)-substituted acridones needed to reverse vinblastine resistance in multidrug resistant (MDR) cancer cells

In an attempt to find clinically useful modulators of multidrug resistance (MDR), a series of 19 N(10)-substituted-2-methoxyacridone analogues has been synthesized. 2-Methoxyacridone and its derivatives (1-19) were synthesized. Compound 1 was prepared by the Ullmann condensation of o-chlorobenzoic acid and p-anisidine followed by cyclization using polyphosphoric acid. This compound undergoes N-alkylation in the presence of phase transfer catalyst (PTC). Stirring of 2-methoxy acridone with 1-bromo-3-chloropropane or 1-bromo-4-chlorobutane in a two-phase system consisting of organic phase (tetrahydrofuran) and 6N potassium hydroxide in the presence of tetrabutylammonium bromide leads to the formation of compounds 2 and 11 in good yield. N-(omega-Chloroalkyl) analogues were found to undergo iodide catalyzed nucleophilic substitution reaction with various secondary amines. Products were characterized by UV, IR, 1H and 13C NMR, mass-spectral data and elemental analysis. The lipophilicity expressed in log(10) P and pK(a) of compounds have been determined. All compounds were examined for their ability to increase the uptake of vinblastine (VLB) in MDR KBCh(R)-8-5 cells and the results showed that the compounds 7, 10, 12, and 15-19 at 100 microM caused a 1.05- to 1.7-fold greater accumulation of vinblastine than did a similar concentration of the standard modulator, verapamil (VRP). However, the effects on VLB uptake were specific because these derivatives had little effect in the parental drug sensitive line KB-3-1. Steady state accumulation of VLB, a substrate for P-glycoprotein (P-gp) mediated efflux, was studied in the MDR cell line KBCh(R)-8-5 in the presence and absence of novel MDR modulators. Results of the efflux experiment showed that VRP and each of the modulators (1-19) significantly inhibited the efflux of VLB, suggesting that they may be competitors for P-gp. From among the compounds examined, 14 except 1, 2, 4, 8, and 11, exhibited greater efflux inhibiting activity than VRP. All the 19 compounds effectively compete with [(3)H] azidopine for binding to P-gp, pointed out this transport membrane protein as their likely site of action. Cytotoxicity has been determined and the IC(50) values lie in the range 8.00-18.50 microM for propyl and 4-15 microM for butyl derivatives against KBCh(R)-8-5 cells suggesting that the antiproliferative activity increases as chain length increases from 3 to 4 carbons at N(10)-position. Compounds at IC(10) were evaluated for their efficacy to modulate the cytotoxicity of VLB in KBCh(R)-8-5 cells and found that the modulators enhanced the cytotoxicity of VLB by 5- to 35-fold. Modulators 12, 14-16, and 19 like VRP, were able to completely reverse the 24-fold resistance of KBCh(R)-8-5 cells to VLB. Examination of the relationship between lipophilicity and antagonism of MDR showed a reasonable correlation suggesting that hydrophobicity is one of the determinants of potency for anti-MDR activity of 2-methoxyacridones.     

Two inositol hexakisphosphate kinases drive inositol pyrophosphate synthesis in plants

Inositol pyrophosphates are unique cellular signaling molecules with recently discovered roles in energy sensing and metabolism. Studies in eukaryotes have revealed that these compounds have a rapid turnover, and thus only small amounts accumulate. Inositol pyrophosphates have not been the subject of investigation in plants even though seeds produce large amounts of their precursor, myo‐inositol hexakisphosphate (InsP₆). Here, we report that Arabidopsis and maize InsP₆ transporter mutants have elevated levels of inositol pyrophosphates in their seed, providing unequivocal identification of their presence in plant tissues. We also show that plant seeds store a little over 1% of their inositol phosphate pool as InsP₇ and InsP₈. Many tissues, including, seed, seedlings, roots and leaves accumulate InsP₇ and InsP₈, thus synthesis is not confined to tissues with high InsP₆. We have identified two highly similar Arabidopsis genes, AtVip1 and AtVip2, which are orthologous to the yeast and mammalian VIP kinases. Both AtVip1 and AtVip2 encode proteins capable of restoring InsP₇ synthesis in yeast mutants, thus AtVip1 and AtVip2 can function as bonafide InsP₆ kinases. AtVip1 and AtVip2 are differentially expressed in plant tissues, suggesting non‐redundant or non‐overlapping functions in plants. These results contribute to our knowledge of inositol phosphate metabolism and will lay a foundation for understanding the role of InsP₇ and InsP₈ in plants.     

Linkage disequilibrium and population-structure analysis among Capsicum annuum L. cultivars for use in association mapping

Knowledge of population structure and linkage disequilibrium among the worldwide collections of peppers currently classified as hot, mild, sweet and ornamental types is indispensable for applying association mapping and genomic selection to improve pepper. The current study aimed to resolve the genetic diversity and relatedness of Capsicum annuum germplasm by use of simple sequence repeat (SSR) loci across all chromosomes in samples collected in 2011 and 2012. The physical distance covered by the entire set of SSRs used was 2,265.9 Mb from the 3.48-Gb hot-pepper genome size. The model-based program STRUCTURE was used to infer five clusters, which was further confirmed by classical molecular-genetic diversity analysis. Mean heterozygosity of various loci was estimated to be 0.15. Linkage disequilibrium (LD) was used to identify 17 LD blocks across various chromosomes with sizes from 0.154 Kb to 126.28 Mb. CAMS-142 of chromosome 1 was significantly associated with both capsaicin (CA) and dihydrocapsaicin (DCA) levels. Further, CAMS-142 was located in an LD block of 98.18 Mb. CAMS-142 amplified bands of 244, 268, 283 and 326 bp. Alleles 268 and 283 bp had positive effects on both CA and DCA levels, with an average R ² of 12.15 % (CA) and 12.3 % (DCA). Eight markers from seven different chromosomes were significantly associated with fruit weight, contributing an average effect of 15 %. CAMS-199, HpmsE082 and CAMS-190 are the three major quantitative trait loci located on chromosomes 8, 9, and 10, respectively, and were associated with fruit weight in samples from both years of the study. This research demonstrates the effectiveness of using genome-wide SSR-based markers to assess features of LD and genetic diversity within C. annuum.