phenylalanine derivatives with small ortho substitutions
were genetically encoded in Escherichia coli and
mammalian cells at an amber codon using a previously reported,
rationally designed pyrrolysyl-tRNA synthetase mutant (PylRS(N346A/C348A))
coupled with tRNACUAPyl. Ortho substitutions of the phenylalanine
derivatives reported herein include three halides, methyl, methoxy,
nitro, and nitrile. These compounds have the potential for use in
multiple biochemical and biophysical applications. Specifically, we
demonstrated that o-cyano-phenylalanine could be
used as a selective sensor to probe the local environment of proteins
and applied this to study protein folding/unfolding. For six of these
compounds this constitutes the first report of their genetic incorporation
in living cells. With these compounds the total number of substrates
available for PylRS(N346A/C348A) is increased to nearly 40, which
demonstrates that PylRS(N346A/C348A) is able to recognize phenylalanine
with a substitution at any side-chain aromatic position as a substrate.
To our knowledge, PylRS(N346A/C348A) is the only aminoacyl-tRNA synthetase
with such a high substrate promiscuity.
Chloroquine was a cheap, extremely effective drug against Plasmodium falciparum until resistance arose. One approach to reversing resistance is the inhibition of chloroquine efflux from its site of action, the parasite digestive vacuole. Chloroquine accumulation studies have traditionally relied on radiolabelled chloroquine, which poses several challenges. There is a need for development of a safe and biologically relevant substitute. We report here a commercially-available green fluorescent chloroquine-BODIPY conjugate, LynxTag-CQGREEN, as a proxy for chloroquine accumulation. This compound localized to the digestive vacuole of the parasite as observed under confocal microscopy, and inhibited growth of chloroquine-sensitive strain 3D7 more extensively than in the resistant strains 7G8 and K1. Microplate reader measurements indicated suppression of LynxTag-CQGREEN efflux after pretreatment of parasites with known reversal agents. Microsomes carrying either sensitive- or resistant-type PfCRT were assayed for uptake; resistant-type PfCRT exhibited increased accumulation of LynxTag-CQGREEN, which was suppressed by pretreatment with known chemosensitizers. Eight laboratory strains and twelve clinical isolates were sequenced for PfCRT and Pgh1 haplotypes previously reported to contribute to drug resistance, and pfmdr1 copy number and chloroquine IC50s were determined. These data were compared with LynxTag-CQGREEN uptake/fluorescence by multiple linear regression to identify genetic correlates of uptake. Uptake of the compound correlated with the logIC50 of chloroquine and, more weakly, a mutation in Pgh1, F1226Y.
Nε-acryloyl-L-lysine, a noncanonical amino acid with an electron deficient olefin, is genetically encoded in Escherichia coli using a pyrrolysyl-tRNA synthetase mutant in coordination with tRNACUAPyl. The acrylamide moiety is stable in cells, whereas it is active enough to perform a diverse set of unique reactions for protein modifications in vitro. These reactions include 1,4-addition, radical polymerization, and 1,3-dipolar cycloaddition. We demonstrate that a protein incorporated with Nε-acryloyl-L-lysine is efficiently modified with thiol-containing nucleophiles at slightly alkali conditions and the acrylamide moiety also allows rapid radical copolymerization of the same protein into a polyacrylamide hydrogel at physiological pH. At physiological conditions, the acrylamide functionality undergoes a fast 1,3-dipolar cycloaddition reaction with diaryl nitrile imine to show turn-on fluorescence. We have used this observation to demonstrate site-specific fluorescent labeling of proteins incorporated with Nε-acryloyl-L-lysine both in vitro and in living cells. This critical development allows easy access to an array of modified proteins for applications where high specificity and reaction efficiency are needed.
Blood-sucking insects use olfactory cues in a variety of behavioral contexts, including host-seeking and aggregation. In triatomines, which are obligated blood-feeders, it has been shown that the response to CO2, a host-associated olfactory cue used almost universally by blood-sucking insects, is modulated by hunger. Host-finding is a particularly dangerous task for these insects, as their hosts are also their potential predators. Here we investigated whether olfactory responses to host-derived volatiles other than CO2 (nonanal, α-pinene and (−)-limonene), attractive odorant mixtures (yeast volatiles), and aggregation pheromones (present in feces) are also modulated by starvation in the blood-sucking bug Rhodnius prolixus. For this, the responses of both non-starved and starved insects were individually tested at the beginning of the scotophase using a dual-choice “T-shaped” olfactometer, in which one of its arms presented odor-laden air and the other arm presented odorless air. We found that the response of non-starved insects towards host-odorants and odorant mixtures was odor-dependent: insects preferred the odor-laden arm of the maze when tested with α-pinene, the odorless arm of the maze when tested with (−)-limonene, and distributed at random when tested with yeast volatiles or nonanal. In contrast, starved insects significantly preferred the odor-laden arm of the maze when tested with host-odorants or yeast volatiles. When tested with aggregation pheromones, non-starved insects distributed randomly between the arms of the maze, while starved insects preferred the odorless arm of the maze; insects that were even more starved (8–9 weeks post-ecdysis) significantly preferred the odor-laden arm of the maze. We postulate that this odor- and starvation-dependent modulation of sensory responses has a high adaptive value, as it minimizes the costs and risks associated with the associated behaviors. The possible physiological mechanisms underlying these modulatory effects are discussed.
triatomine; Rhodnius prolixus; olfaction; behavior; sensory modulation
Plasmodium falciparum is the etiological agent of malignant malaria and has been shown to exhibit features resembling programmed cell death. This is triggered upon treatment with low micromolar doses of chloroquine or other lysosomotrophic compounds and is associated with leakage of the digestive vacuole contents. In order to exploit this cell death pathway, we developed a high-content screening method to select compounds that can disrupt the parasite vacuole, as measured by the leakage of intravacuolar Ca2+. This assay uses the ImageStream 100, an imaging-capable flow cytometer, to assess the distribution of the fluorescent calcium probe Fluo-4. We obtained two hits from a small library of 25 test compounds, quinacrine and 3′,4′-dichlorobenzamil. The ability of these compounds to permeabilize the digestive vacuole in laboratory strains and clinical isolates was validated by confocal microscopy. The hits could induce programmed cell death features in both chloroquine-sensitive and -resistant laboratory strains. Quinacrine was effective at inhibiting field isolates in a 48-h reinvasion assay regardless of artemisinin clearance status. We therefore present as proof of concept a phenotypic screening method with the potential to provide mechanistic insights to the activity of antimalarial drugs.
Cytosine-guanine oligodeoxynucleotide (CpG ODN) motifs of bacterial DNA are recognized through toll-like receptor 9 (TLR9) and are potent activators of innate immunity. However, the interaction between TLR9 and CpG ODN in aquatic species has not been well characterized. Hence, cobia TLR9 isoform B (RCTLR9B) was cloned and its expression and induction in intestine were investigated. RCTLR9B cDNA consists of 3113bp encoding 1009 amino acids containing three regions, leucine rich repeats, transmembrane domain, and toll/interleukin-1 receptor (TIR) domain. Intraperitoneal injection of CpG ODN 2395 upregulated RCTLR9 A and B and MyD88 and also induced the expressions of Mx, chemokine CC, and interleukin IL-1β. Cobia intraperitoneally injected with CpG ODN 1668 and 2395 had increased survival rates after challenge with Photobacterium damselae subsp. piscicida. In addition, formulation of CpG ODN with formalin-killed bacteria (FKB) and aluminum hydroxide gel significantly increased expressions of RCTLR9 A (50 folds) and B (30 folds) isoforms at 10 dpi (CpG ODN 1668) and MyD88 (21 folds) at 6 dpv (CpG ODN 2395). Subsequently, IL-1β increased at 6 dpv in 1668 group. No histopathological damage and inflammatory responses were observed in the injected cobia. Altogether, these results facilitate CpG ODNs as an adjuvant to increase bacterial disease resistance and efficacy of vaccines in cobia.
Three new eunicellin-type diterpenoids, krempfielins N–P (1–3), were isolated from a Taiwanese soft coral Cladiella krempfi. The structures of the new metabolites were elucidated by extensive spectroscopic analysis and by comparison with spectroscopic data of related known compounds. Compound 3 exhibited activity to inhibit superoxide anion generation. Both 1 and3, in particular 1, were shown to display significant anti-inflammatory activity by inhibiting the elastase release in FMLP/CB-induced human neutrophils.
Cladiella krempfi; eunicellin-type diterpenoid; anti-inflammatory agent; elastase
Acute otitis media and otitis media with effusion are common childhood disorders, a source of significant morbidity, and a leading cause of antibiotic prescription in primary health care. Although effective treatments are available, some shortcomings remain, and thus better treatments would be welcome. Recent discoveries within the field of otitis media research relating to its etiology and pathogenesis have led to further investigation aimed at developing novel treatments. This article provides a review of the latest evidence relating to the understanding of acute otitis media and otitis media with effusion, current treatment strategies, their limitations, new areas of research, and novel strategies for treatment.
otitis media; ear; hearing; infection; biofilm; antibiotics
New four eunicellin-based diterpenoids, krempfielins J–M (1–4) were isolated from the organic extract of a Taiwanese soft coral Cladiella krempfi. The structures of the new metabolites were elucidated on the basis of extensive spectroscopic analysis. The structure of compound 2 is rare due to the presence of the highly oxygenated pattern. Anti-inflammatory activity of 1–6 to inhibit the superoxide anion generation and elastase release in FMLP/CB-induced human neutrophils was also evaluated, and 2 and 4 were shown to possess the ability to inhibit the elastase release.
Cladiella krempfi; eunicellin-based diterpenoid; elastase; anti-inflammatory agent
Cancer is one of the deadliest diseases against humans. To tackle this menace, humans have developed several high-technology therapies, such as chemotherapy, tomotherapy, targeted therapy, and antibody therapy. However, all these therapies have their own adverse side effects. Therefore, recent years have seen increased attention being given to the natural food for complementary therapy, which have less side effects. Garlic (Dà Suàn; Allium sativum), is one of most powerful food used in many of the civilizations for both culinary and medicinal purpose. In general, these foods induce cancer cell death by apoptosis, autophagy, or necrosis. Studies have discussed how natural food factors regulate cell survival or death by autophagy in cancer cells. From many literature reviews, garlic could not only induce apoptosis but also autophagy in cancer cells. Autophagy, which is called type-II programmed cell death, provides new strategy in cancer therapy. In conclusion, we wish that garlic could be the pioneer food of complementary therapy in clinical cancer treatment and increase the life quality of cancer patients.
Autophagy; Cancer; Complementary therapy; Garlic
The antihepatoma activity and liver protective function of the fermentation products (5 L fermenator) of Ganoderma lucidum (GL; 靈芝 Ling Zhi) cultivated in a medium containing black soybean (BS; 黑豆 Hēi Dòu) and Astragalus membranaceus (AM; 生黃耆 Shēng Huáng Qí) at different fermentation temperatures were investigated in this study. Hep 3B cells pretreated with lovastatin were used to study the antihepatoma activity, and possible active components were analyzed by reverse-phase high-performance liquid chromatography. Carbon tetrachloride (CCl4)-induced primary rat hepatocyte injury was further used to evaluate the liver protective activity of the fermentation products. While all the GL broth filtrates do not inhibit the growth of Hep 3B cells, the ethanolic extract from GL-2 mycelia (GL-2-mE), cultivated in the medium containing BS (50 g/L) and AM (20 g/L) at 24°C for 11 days showed the best antihepatoma activity (IC50 26.6 μg/mL) than the other ethanolic extracts from GL mycelia, GL fruiting body, BS, and AM did. The antihepatoma activities were correlated with some unknown active components in these samples. Furthermore, GL-2-mE (100 μg/mL) without harmful effect on the growth of normal primary rat hepatocytes significantly maintained cell viability, reduced lactate dehydrogenase leakage, lowered lipid peroxidation, and increased glutathione peroxidase and glutathione S-transferase activities in the CCl4-induced damaged primary rat hepatocytes.
Antihepatoma activity; Astragalus membranaceus; Black soybean; Fermentation; Ganoderma lucidum; Liver protective function
Glioma is still one of the most complicated forms of brain tumor to remove completely due to its location and the lack of an efficient means to specifically eliminate tumor cells. For these reasons, this study has examined the effectiveness of a nonviral gene therapy approach utilizing a tumor-selective killer gene on a brain tumor xenograft model.
Methods and results
The therapeutic apoptin gene was recombined into the JDK plasmid and delivered into human brain tumor cells (U87MG) by using a polyamidoamine dendrimer with an arginine surface (PAM-RG4). Studies in vitro showed that the PAM-RG4/apoptin plasmid polyplex exhibited a particularly high transfection activity of .40%. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, 4′,6-Diamidino-2-phenylindole (DAPI) TUNEL assay, DAPI staining, and caspase-3 activity assay verified that the tumor cells had undergone apoptosis induced by apoptin. For in vivo studies, the polyplex was injected into tumors, which were induced by injecting U87MG cells intradermally into nude mice. Based on hematoxylin and eosin staining, epidermal growth factor receptor immunohistochemistry results and tumor volume measurement results, tumor growth was effectively inhibited and no specific edema, irritation, or other harm to the skin was observed after polyplex injection. The in vivo expression of apoptin and the induction of apoptosis were verified by reverse-transcription polymerase chain reaction analysis, TUNEL assay, and DAPI staining.
The PAM-RG4/apoptin gene polyplex is a strong candidate for brain tumor therapeutics because of the synergistic effect of the carrier’s high transfection efficiency (35%–40%) in glioma cells and the selective apoptosis-inducing activity of apoptin in tumor cells.
apoptin; PAM-RG4; malignant glioma; nonviral gene therapy; apoptosis
According to the prediction of the 2008 World Health Organization (WHO) report, depression will be the highest burden disease by the year 2030. Daylily flower (金針花 Jīn Zhēn Huā ; the flower of Hemerocallis fulva) is traditionally used for soothing in Chinese dietary therapy. The major flavonoid of daylily flowers, rutin, is also characterized to be an antidepressant. In this study, we investigated the antidepressant effects of ethanol extract of daylily flowers (DFEtoH) and rutin by forced swimming test (FST) and neurotransmitter metabolism of brain regions (frontal cortex, hippocampus, striatum, and amygdala). Results show that either short- or long-term tests, the extract and rutin significantly reduce the immobility time and increased swimming time of FST, which are compared with the vehicle (P < 0.05). The extract and rutin also increase the serotonin, norepinephrine, and dopamine concentration of these brain regions (P < 0.05). In long-term tests, the daylily flowers extract markedly increased serotonin concentration and reduced serotonin turnover rate in these brain regions but not frontal cortex. In conclusion, present data illustrated that DFEtoH does have antidepressant-like effects possibly via the regulation of serotonergic system. Moreover, rutin might be playing a very important role in the antidepressant-like effects of DFEtoH.
Antidepressant; Daylily; Forced swimming test; Hemerocallis fulva; Serotonergic system
Chinese herbal medicine (中草藥) attracts much attention in the treatment of liver injuries. Numerous studies have revealed various biological activities of medicinal mushrooms such as Antrodia Cinnamomea (牛樟芝). Although A. cinnamomea is rare in the wild, recent developments in fermentation and cultivation technologies make the mycelia and fruiting bodies of this valuable medicinal mushroom readily available. Liver diseases such as fatty liver, hepatitis, hepatic fibrosis, and liver cancer are complicated processes of liver injuries that have tremendous impact on human society. In this article, we reviewed studies about the hepatoprotective effects of the fruiting bodies and mycelia of A. cinnamomea performed in different experimental models. The results of those studies suggest the potential application of A. cinnamomea in preventing and treating liver diseases and its potential to be developed into health foods or new drugs.
Antrodia Cinnamomea; Fruiting bodies; Mycelia; Liver injury
Antrodia cinnamomea, a Taiwan-specific medicinal mushroom, can manipulate biological activities, including hepatoprotection, anti-inflammation, anti-hepatitis B virus activity, anticancer activity, etc. In this study, the anti-liver cancer activity and molecular mechanisms of eburicoic acid, the second most abundant triterpenoid from the fruiting bodies of basswood cultivated Antrodia cinnamomea was investigated using the human hepatoma Hep 3B cells. The results show that eburicoic acid effectively reduced Hep 3B cell viability within 24 hours, and the IC50 was 18.4 μM, which was equivalent to 8.7 μg/mL. Besides, eburicoic acid induced conversion of LC3-I to LC3-II and a large number of autophagosomes/autolysosomes formation. In depth investigation for the molecular mechanisms, revealed that eburicoic acid firstly promoted reactive oxygen species generation and ATP depletion, leading to endoplasmic reticulum stress, followed by elevated cytosolic calcium ion concentration and BiP expression, downregulated phosphorylation of DAPK, upregulated phosphorylation of Beclin-1, JNK, and Bcl-2, and finally induced autophagy in Hep 3B cells. These results indicate that eburicoic acid has significant anti-liver cancer effects and more distinctive mechanisms.
Liver cancer; Antrodia cinnamomea; Eburicoic acid; Autophagy; ER stress
The effect of allicin, an active ingredient of garlic, on lactate dehydrogenase (LDH) leakage, lipid peroxidation, glutathione (GSH) content, and GSH-related enzyme activity was investigated in primary hepatocytes. In this study, allicin was synthesized in our laboratory as an experimental material, and primary hepatocytes isolated from Sprague-Dawley rats were used as an experimental model. According to the results, hepatocytes treated with 10 μM allicin did not differ from the control on LDH leakage during various incubation times. When the hepatocytes were treated with 10 μM allicin, their levels of thiobarbituric acid reactive-substances (TBARS) did not differ significantly from that of the control within the 8-h incubation. However, the TBARS values of hepatocytes treated with 30 and 50 μM allicin were higher compared to the control after incubation for 4 h and 8 h, respectively. The hepatocyte intracellular GSH content was significantly higher than that of the control after 30 μM allicin treatment, but treatment with 50 μM allicin caused a significant GSH depletion after incubation for 4 h or longer. In addition, when hepatocytes were treated for 24 h with 10 or 30 μM allicin, glutathione peroxidase (GPx) activity was significantly increased compared to that of the control, whereas 50 μM allicin treatment for 24 h or longer significantly decreased the GPx activity. Glutathione reductase (GRd) activity was significantly increased when the hepatocytes were treated with 10 μM allicin for 24 h, but GRd activity significantly decreased when the hepatocytes were treated with 50 μM allicin. However, hepatocytes treated for 24 h with 10 or 30 μM allicin showed significantly increased glutathione S-transferase (GST) activity compared to the control. These results suggest that 10 μM allicin potentially enhances the antioxidation and detoxification capabilities of primary rat hepatocytes.
allicin; antioxidation; detoxification; primary rat hepatocytes
Sexual reproduction of scleractinian coral is among the most important means of establishing coral populations. However, thus far, little is known about the mechanisms underlying coral gametogenesis. To better understand coral germ cell development, we performed a histological analysis of gametogenesis in Euphyllia ancora and characterized the coral homolog of the Drosophila germline marker gene vasa. The histological analysis revealed that E. ancora gametogenesis occurs in the mesenterial mesoglea between the mesenterial filaments and the retractor muscle bands. The development of germ cells takes approximately one year in females and half a year in males. Staining of tissue sections with an antibody against E. ancora Vasa (Eavas) revealed anti-Eavas immunoreactivity in the oogonia, early oocyte, and developing oocyte, but only faint or undetectable reactivity in developing oocytes that were >150 µm in diameters. In males, Eavas could be detected in the spermatogonia and primary spermatocytes but was only faintly detectable in the secondary spermatocytes, spermatids, and sperms. Furthermore, a reverse transcription-polymerase chain reaction analysis and Western blotting analysis of unfertilized mature eggs proved the presence of Eavas transcripts and proteins, suggesting that Eavas may be a maternal factor. Vasa may represent a germ cell marker for corals, and would allow us to distinguish germ cells from somatic cells in coral bodies that have no distinct organs.
Garlic (大蒜 dà suàn; the bulb of Allium sativum), bestowed with an array of organosulfur compounds finds its application in treating many ailments including cardiovascular problems, common cold, bacterial and fungal infections and cancer. Numerous epidemiological evidences document the beneficial effects of various bioactive organosulfur compounds of garlic against different types of cancer. Studies involving the animal and cell models indicate garlic bioactive compounds could be effective in treating all the stages of cancer. This review gives an update on the recent pre-clinical and clinical trials, carried out to evaluate the efficacy of various garlic bioactive compounds along with the mechanism of action pertaining to major digestive cancers including liver, gastric and colorectal cancers. The major anti-carcinogenic mechanisms are caspase dependent and/or independent induction of apoptosis, anti-proliferative, anti-metastasis, anti-oxidant and immunomodulative properties. Form the clinical trials an increase in the garlic consumption of 20 g/day reduced the risk of gastric and colorectal cancer. In summary, increased uptake of garlic in diet may prevent the incidence of digestive cancers.
Liver cancer; Gastric cancer; Colorectal cancer; Garlic; Anticarcinogenic agent
Alcoholic liver disease (ALD) is a complex chronic disease and is associated with a spectrum of liver injury ranging from steatosis and steatohepatitis to fibrosis and cirrhosis. Since effective therapies for ALD are still limited, Chinese herbal medicine is thought to be an important and alternative approach. This review focuses on the current scientific evidence of ALD by ten Chinese Materia Medica (中藥 zhōng yào), including Salviae Miltiorrhizae Radix (丹參 dān shēn), Notoginseng Radix (三七 sān qī), Lycii Fructus (枸杞子 gǒu qǐ zǐ), Cnidii Fructus (蛇床子 shé chuáng zǐ), Gentianae Radix (龍膽 lóng dǎn), Puerariae Radix (葛根 gé gēn), Puerariae Flos (葛花 gé huā), Magnoliae Officinalis Cortex (厚朴 hòu pò), Platycodonis Radix (桔梗 jié gěng), and Trigonellae Semen (胡蘆巴 hú lú bā). Potential mechanisms of these herbal medicines in ALD are involved in amelioration of enhanced inflammation, reduction of hepatic oxidative stress and lipogenesis, and enhancement of intestinal permeability in alcohol-induced liver injury models in vitro and in vivo. Accordingly, the evidenced therapeutic potential suggests that these herbs are promising candidates for prevention and development of new drugs for ALD in the future.
Alcoholic liver disease; Alcohol-induced liver injury; Herbal medicine; Chinese Materia Medica; Traditional Chinese medicine
Oxidative stress and inflammatory condition has been broadly accepted being associated with the progression of diabetes. On the other hand, garlic (大蒜 dà suàn, bulb of Allium sativum) has been shown to possess both antioxidant and anti-inflammatory action in several clinical conditions. Our previous study demonstrated that treatment with garlic oil improves oral glucose tolerance and insulin tolerance and improves the insulin-stimulated utilization of glucose to synthesize glycogen in skeletal muscle in streptozotocin (STZ)-induced diabetes, in vivo and ex vivo, respectively. The aim of the present study is to investigate the antioxidant and anti-inflammatory effects of garlic oil (GO) in the skeletal muscle of diabetic rats. Rats with STZ-induced diabetes received GO (10, 50, or 100 mg/kg body weight) or corn oil by gavage every other day for 3 weeks. Control rats received corn oil only. GO dose-dependently improved insulin sensitivity, as assessed by the insulin tolerance test, and oral glucose tolerance. GO significantly elevated total glutathione and glutathione peroxidase activity and lowered the nitrate/nitrite content in skeletal muscle at 50 and 100 mg/kg and significantly elevated glutathione reductase activity and lowered lipid peroxidation at 100 mg/kg. By contrast, GO did not reverse diabetes-induced elevation of IL-1β and TNF-α in skeletal muscle at any tested dose. On the other hand, GO elevated the expression of GLUT4 in skeletal muscle along with glycogen content as observed with PAS staining. In conclusion, the antidiabetic effect of garlic oil is associated with ameliorated oxidative stress in skeletal muscle.
Antidiabetic; Antioxidant; Garlic oil; Skeletal muscle; Streptozotocin
Hepatoma is a leading cause of death in the world. SK-Hep-1 and HA22T/VGH cells are poorly differentiated human hepatocellular carcinoma cell lines with invasive and migratory abilities. Agaricus blazei (AB) is a mushroom with many biological effects and active ingredients, and the ethanolic extract of AB fermentation product (AB-pE) was demonstrated to inhibit the growth of hepatoma Hep3B and HepG2 cells in our previous study. In this study, we further investigated the anticancer and anti-invasive abctivities of the AB-pE. Results showed that the AB-pE inhibited the growth of SK-Hep1 and HA22T/VGH cells (with IC50 values of 26.8 and 28.7 μg/mL, respectively) and led cells toward apoptosis after 48 h of treatment. Activation of caspase-3 by AB-pE (12.5~200 μg/mL) in a dose-dependent manner was observed in both cell lines using fluorescence microscopy and flow cytometry. The apoptosis triggered by the AB-pE was regulated by the increased expression of Bax, the activation of caspase-3, caspase-9, and PARP, and the decreased expression of Bcl-2. Additionally, the AB-pE showed the potential ability to inhibit invasion of SK-Hep1 and HA22T/VGH cells according to the results of a Matrigel invasion assay. Our results suggested that the AB-pE may be a further developed for its potential against hepatoma due to its antiproliferative (via apoptosis) and anti-invasive activities in hepatoma cells.
Agaricus blazei; Anti-hepatoma activity; Antitumor invasion activity; SK-Hep-1 cells; HA22T/VGH cells
High-dose tissue-specific delivery of therapeutic agents would be a valuable clinical strategy. We have previously shown that repeated transcranial focused ultrasound is able to increase the delivery of Evans blue significantly into brain tissue. The present study shows that repeated pulsed high-intensity focused ultrasound (HIFU) can be used to deliver high-dose atherosclerotic plaque-specific peptide-1 (AP-1)-conjugated liposomes selectively to brain tumors.
Firefly luciferase (Fluc)-labeled human GBM8401 glioma cells were implanted into NOD-scid mice. AP-1-conjugated liposomal doxorubicin or liposomal doxorubicin alone was administered followed by pulsed HIFU and the doxorubicin concentration in the treated brains quantified by fluorometer. Growth of the labeled glioma cells was monitored through noninvasive bioluminescence imaging and finally the brain tissue was histologically examined after sacrifice.
Compared with the control group, the animals treated with 5 mg/kg injections of AP-1 liposomal doxorubicin or untargeted liposomal doxorubicin followed by repeated pulsed HIFU not only showed significantly enhanced accumulation of drug at the sonicated tumor site but also a significantly elevated tumor-to-normal brain drug ratio (P < 0.001). Combining repeated pulsed HIFU with AP-1 liposomal doxorubicin or untargeted liposomal doxorubicin has similar antitumor effects.
This study demonstrates that targeted or untargeted liposomal doxorubicin, followed by repeated pulsed HIFU, is a promising high-dose chemotherapy method that allows the desired brain tumor region to be targeted specifically.
repeated focused ultrasound; interleukin-4 receptor; blood-brain barrier; brain tumor; target drug delivery
The catadromous Platyeriocheir formosa is a crab endemic in Taiwan. To conserve P. formosa population diversity and ensure the sustainable use of this natural resource, we have developed new genetic markers, 17 polymorphic microsatellite loci, to promote the study of its population genetics in the future. In this study, more than 70 microsatellite sequences were found. Among these, 18 loci were selected to analyze the genetic diversity of P. formosa. With the exception of the Pfo15 locus, all of the remaining loci were polymorphic with allelic numbers ranging from 3–14. Heterozygosity within all 17 polymorphic loci ranged from 0.2–0.95 with an average of 0.55, which suggested that these loci are proper markers for studying population genetics. After we tested cross-specific amplification, eight and six primer sets could be successfully used for the amplification of microsatellite loci in morphologically similar Eriocheir sinensis and E. japonica, respectively; this suggests that they are useful markers for closely related species.
catadromous; crab; cross-specific amplification; polymorphic loci