Association between TAP1 gene polymorphisms and alopecia areata in a Korean population.

The transporter 1 ATP-binding cassette sub-family B (MDR/TAP) gene (TAP1) is located in the major histocompatibility complex class II region, and forms a heterodimer that plays a key role in endogenous antigen presentation pathways. Investigation of polymorphisms identified in these loci has revealed an association with several autoimmune disorders. Alopecia areata (AA) is a common autoimmune disease resulting from T cell-induced damage to hair follicles. The present study documents for the first time a comparison between the allelic and genotypic frequencies of TAP1 single nucleotide polymorphisms (SNPs) in patients with AA and those of a control group, using a direct sequencing method. Our results suggest an association between a promoter SNP (rs2071480) and susceptibility to this disease.

Characterization of novel microsatellite markers derived from Korean rose bitterling (Rhodeus uyekii) genomic library.

Korean rose bitterling (Rhodeus uyekii) is a freshwater fish endemic to Korea. Natural populations of this species have experienced severe declines as a result of habitat fragmentation and water pollution. To conserve and restore R. uyekii, the genetic diversity of this species needs to be assessed at the population level. Eighteen novel polymorphic microsatellite loci for R. uyekii were developed using an enriched partial genomic library. Polymorphisms at these loci were studied in 150 individuals collected from three populations. The number of alleles at each locus ranged from 3 to 47 (mean = 17.1). Within the populations, the observed heterozygosity ranged from 0.032 to 1.000, expected heterozygosity from 0.082 to 0.967, and polymorphism information content from 0.078 to 0.950. Six loci showed significant deviation from Hardy-Weinberg equilibrium after Bonferroni's correction, and no significant linkage disequilibrium was detected between most locus pairs, except in three cases. These highly informative microsatellite markers should be useful for genetic population structure analyses of R. uyekii.

Hyaluronidase treatment of acute lymphedema in a mouse tail model.

The purpose of this study was to investigate the impact of hyaluronidase (HAase) on lymphedema using an acute mouse tail lymphedema model. Six-week-old mice served to produce acute lymphedema and were then either treated with HAase injection or used as operative controls. An additional group of unmanipulated normal mice was used for comparison. Tail volumes were measured for 23 days and histological changes examined. Western blot analysis was conducted to quantify lymphatic vessel endothelial hyaluronan receptor (LYVE)-1, tumor necrosis factor (TNF)-alpha, transforming growth factor (TGF)-beta1, podoplanin, CD 44, and vascular endothelial growth factor receptor3 (VEGFR3) expression levels. The operative control group showed an increase in thickness of the dermis and subdermis, microlymphatic dilatation, and an increase in neutrophils. In contrast, the HAase treated group exhibited alleviation of inflammation evidenced by a decline in microlymphatic dilatation and neutrophils and an overall increase in microlymphatic vessels. Western blot analysis demonstrated that TNF-alpha and TGF-beta1 expression declined but CD44 expression increased in the HAase treated group. Levels of LYVE1, podoplanin, and VEGFR3 also increased significantly in the HAase group. Our results indicate that HAase treatment in the acute mouse tail model reduced lymphedema volume possibly through degradation of HA trafficking, which reduced inflammation and fibrosis in tissues and stimulated lymphangiogenesis.

Coriandrum sativum L. protects human keratinocytes from oxidative stress by regulating oxidative defense systems.

BACKGROUND: Oxidative radicals are major environmental causes of human skin damage. Oxidative defense factors, including nuclear factor erythroid-derived 2-related factor 2 (Nrf2), are centrally involved in repairing skin cells or protecting them from oxidative damage. Coriandrum sativum L. (coriander; CS) is a commonly consumed food and a traditional phytomedicine in Asia and Europe. In this study, we examined the protective effects of a standardized CS leaf extract against oxidative stress in human HaCaT keratinocytes. METHODS AND RESULTS: CS significantly and dose-dependently protected cells against reduced cell viability caused by H2O2-induced damage, as assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Other assays demonstrated that CS protected HaCaT cells by increasing the levels of glutathione and activities of oxidative defense enzymes, such as superoxide dismutase and catalase. Moreover, it increased the expression of activated Nrf2, which plays a crucial role in protecting skin cells against oxidative stress. CONCLUSION: These results suggest that CS protects human keratinocytes from H2O2-induced oxidative stress through antioxidant effects.

Adenovirus-relaxin gene therapy for keloids: implication for reversing pathological fibrosis.

BACKGROUND: Keloids or hypertrophic scars are pathological proliferations of the dermal skin layer resulting from excessive collagen deposition. Because the hormone relaxin (RLX) inhibits collagen synthesis and expression in stimulated fibroblasts, an adenovirus expressing RLX (dE1-RGD/lacZ/RLX) was generated. OBJECTIVES: To investigate the effect of RLX-expressing adenovirus on expression of various extracellular matrix (ECM) components in primary keloid spheroids. METHODS: The expression levels of type I and III collagen, fibronectin and elastin were investigated by immunohistochemistry in primary keloid spheroids transduced with the RLX-expressing adenovirus. RESULTS: Immunohistochemical analysis showed that expression of major ECM components (e.g. type I and III collagen, elastin and fibronectin) was markedly reduced in primary keloid spheroids transduced with dE1-RGD/lacZ/RLX. CONCLUSIONS: These results suggest that the antifibrotic effect of RLX-expressing adenovirus may have therapeutic effects on keloids by reversing pathological fibrosis and preventing keloid recurrence after surgical excision.

Usefulness of direct sequencing of pooled DNA for SNP identification and allele-frequency determination compatible with a common disease/common variant hypothesis.

We examined the efficiency of direct sequencing of pooled DNA for developing common single nucleotide polymorphisms (SNPs) and its accuracy for estimating allele frequencies. A pool of 200 control DNAs was established and was used for developing SNPs and estimating minor allele frequencies (MAF). The sensitivity of the pooled DNA method for successfully detecting an SNP with an MAF >0.01 listed in the database was approximately 0.7; it was particularly efficient for detecting SNPs with MAF >0.1, which is compatible with the common disease/common variant hypothesis. The mean difference between the estimated and the observed MAFs was 0.03 +/- 0.023. The pooled DNA method identified four additional SNPs, for which the allele frequency information was not available in the database. The pooled DNA method is a cost- and time-effective tool for both qualifying and quantifying SNPs with considerable accuracy, and it can be particularly useful for dissecting the common disease/common variant hypothesis; this represents a best-case scenario for large-scale association mapping.

Population structure of the olive flounder (Paralichthys olivaceus) in Korea inferred from microsatellite marker analysis.

The population structure of olive flounder Paralichthys olivaceus was estimated using nine polymorphic microsatellite (MS) loci in 459 individuals collected from eight populations, including five wild and three hatchery populations in Korea. Genetic variation in hatchery (mean number of alleles per locus, A = 10.2-12.1; allelic richness, A(R) = 9.3-10.1; observed heterozygosity, H(O) = 0.766-0.805) and wild (mean number of alleles per locus, A = 11.8-19.6; allelic richness, A(R) = 10.9-16.1; observed heterozygosity, H(O) = 0.820-0.888) samples did not differ significantly, suggesting a sufficient level of genetic variation in these well-managed hatchery populations, which have not lost a substantial amount of genetic diversity. Neighbour-joining tree and principal component analyses showed that genetic separation between eastern and pooled western and southern wild populations in Korea was probably influenced by restricted gene flow between regional populations due to the barrier effects of sea currents. The pooled western and southern populations are genetically close, perhaps because larval dispersal may depend on warm currents. One wild population (sample from Wando) was genetically divergent from the main distribution, but it was genetically close to hatchery populations, indicating that the genetic composition of the studied populations may be affected by hydrographic conditions and the release of fish stocks. The estimated genetic population structure and potential applications of MS markers may aid in the proper management of P. olivaceus populations.

Effect of traditionally used herb Pedalium murex L. and its active compound pedalitin on urease expression - For the management of kidney stone.

Pedalium murex L. is a medicinal herb that has been used for the treatment of diseases related to kidney in the traditional system of medicine. The current study aims to study the effect of ethyl acetate extract of P. murex (EAEP) and its fractionated compound pedalitin against urease production and UreC gene expression in Proteus mirabilis. The selected reference strain Proteus mirabilis (MTCC 425) and the isolates culture of Proteus mirabilis were subjected to study the antibacterial efficacy of P. murex. Expression analysis of P. mirabilis urease gene was successfully done by QPCR. The ethyl acetate extract effectively inhibit the reference Proteus mirabilis and bacterial isolates of Proteus mirabilis in the clinical samples studied. EAEP has showed more potent activity (56.7%) against urease enzyme and pedalitin also exhibited potent activity (30.1%). Using qPCR, the expression of UreC gene of P. mirabilis was controlled by EAEP and also its bioactive compound pedalitin. The present study clearly demonstrated the potency of P. murex in controlling the growth of pathogenic P. mirabilis and to control the expression of urease enzyme production as well as to restrict the urease gene expression in P. mirabilis.

In vitro antioxidant and cytotoxic activities of polyherbal extracts from Vetiveria zizanioides, Trichosanthes cucumerina, and Mollugo cerviana on HeLa and MCF-7 cell lines.

Various metabolites exist in the medicinal plants have lot of potential to cure various diseases and disorders. Plants such as, Vetiveria zizanioides, Trichosanthes cucumerina, and Mollugo cerviana were collected from Western Ghats, Tamilnadu, India. Phytochemicals were extracted from these plants using various organic solvents and tested against Gram-positive and Gram-negative bacteria. The phytochemicals such as, carbohydrate, alkaloids, steroids, saponins, flavonoids and tannin were detected from these medicinal plants. Among the extracts, methanol showed potent activity and this solvent was used to extract polyherbal medicinal plants. Methanol extract of V. zizanioides was found to be highly active against E. coli (27 ± 2 mm), P. mirabilis (19 ± 3 mm) and B. subtilis (18 ± 2 mm). Ethyl acetate extract showed high activity against E. coli (24 ± 2 mm), P. mirabilis (22 ± 3 mm) and B. subtilis (20 ± 1 mm). These three plants were taken at 1:1:1 ratio and extracted with methanol at 1:10 ratio and synergistic activity was tested against bacterial pathogens. Synergistic activity of polyherbal extract was analyzed. The extracted crude herbal medicine was found to be effective against Staphylococcus aureus, E. coli, Enterbacter sp., Pseudomonas aeruginosa, Bacillus subtilis and Proteus mirabilis. The zone of inhibition was 33 ± 3 mm, 17 ± 2 mm, 22 ± 2 mm, 40 ± 2 mm, 33 ± 1 mm and 38 ± 2 mm zone of inhibition against E. coli, S. aureus, P. aeruginosa, P. mirabilis, B. subtilis and Enterobacter sp. Polyherbal extract was found to be highly effective against P. mirabilis and Enterobacter sp. MIC values of polyherbal extract ranged from 29 ± 2.5 µg/ml to 34 ± 2.5 µg/ml. MIC value was found to be less against P. mirabilis and was high against S. aureus. Antioxidant property varied between 49 ± 3% and 95.3 ± 2%. At 20 µg/ml antioxidant activity was reported as 49 ± 3% and it was increased at higher concentrations of polyherbal extract. Two cell lines (HeLa and MCF cell lines) were selected to analyze cytotoxic activity of polyherbal extract. The methanol extract of polyherbal fraction showed cytotoxicity against these two cell lines. The LC50 value was 467 ± 2.9 µg/ml against HeLa cell line and >800 µg/ml against MCF-7 cell lines. The polyherbal extract showed antibacterial, antioxidant and anticancer activities.

Therapeutic efficacy of the root tubers of Aconitum heterophyllum and its substitute Cyperus rotundus in the amelioration of pylorus ligation induced ulcerogenic and oxidative damage in rats.

The phenomenal increase in the demand of herbal drugs, leads to over exploitation of medicinal plants which ultimately resulted in the scarcity and endangerment of many valuable plant species. On observing the difficulties in procuring genuine herbal drugs arose the concept of substitution which was documented in many classical Ayurvedic texts. The present study made a comparative evaluation of the gastroprotective potential of hydroalcoholic extracts of an original drug Aconitum heterophyllum (HAAH) and its substitute Cyperus rotundus (HACR) in the treatment of gastric ulcer under in vivo experimental conditions. The anti-ulcer property of the plant extracts was investigated against pylorus ligation induced ulcer in Wistar albino rats. The results confirmed that both A. heterophyllum and C. rotundus deliver comparable significant protection against gastric ulcer, indicated by a decrease in the free and total acidity, volume of gastric content, total proteins and increase in pH of gastric content, total carbohydrates and total carbohydrates to total proteins ratio. The observed anti-ulcer potential of both the drugs is attributed mainly to prevention of the generation of damaging free radical cascades and oxidant radical release.

SENP1 mediates TNF-induced desumoylation and cytoplasmic translocation of HIPK1 to enhance ASK1-dependent apoptosis.

We have previously shown that tumor necrosis factor (TNF)-induced desumoylation and subsequent cytoplasmic translocation of HIPK1 are critical for ASK1-JNK activation. However, the mechanism by which TNF induces desumoylation of HIPK1 is unclear. Here, we show that SENP1, a SUMO-specific protease, specifically deconjugates SUMO from HIPK1 in vitro and in vivo. In resting endothelial cells (ECs), SENP1 is localized in the cytoplasm where it is complexed with an antioxidant protein thioredoxin. TNF induces the release of SENP1 from thioredoxin as well as nuclear translocation of SENP1. TNF-induced SENP1 nuclear translocation is specifically blocked by antioxidants such as N-acetyl-cysteine, suggesting that TNF-induced translocation of SENP1 is ROS dependent. TNF-induced nuclear import of SENP1 kinetically correlates with HIPK1 desumoylation and cytoplasmic translocation. Furthermore, the wild-type form of SENP1 enhances, whereas the catalytic-inactive mutant form or siRNA of SENP1 blocks, TNF-induced desumoylation and cytoplasmic translocation of HIPK1 as well as TNF-induced ASK1-JNK activation. More importantly, these critical functions of SENP1 in TNF signaling were further confirmed in mouse embryonic fibroblast cells derived from SENP1-knockout mice. We conclude that SENP1 mediates TNF-induced desumoylation and translocation of HIPK1, leading to an enhanced ASK1-dependent apoptosis.

Cloning of profilin (FcPFN) from the shrimp Fenneropenaeus chinensis, a highly expressed protein in white spot syndrome virus (WSSV)-infected shrimp.

We isolated and characterized the profilin (FcPFN) cDNA from hemocytes of Fenneropenaeus chinensis, a unique shrimp species from the Yellow Sea. The FcPFN cDNA consists of 830 bp and encodes a polypeptide of 125 amino acids, having a predicted isoelectric point of 5.06. The deduced amino acid sequence of FcPFN shows 36% and 90% amino acid sequence identity to the profilin genes of Pacific white shrimp Litopenaeus vannamei and black tiger shrimp Penaeus monodon, respectively. The FcPFN mRNA was highly expressed in hemocytes and hepatopancreas and moderately in muscle of normal shrimp. The higher expression of FcPFN mRNA is observed in shrimp infected with the white spot syndrome virus (WSSV), which is a major concern in all shrimp-growing regions of the world. These results suggest a potential role for FcPFN in viral host defense mechanisms.

The antifibrotic potential of a sustained release formulation of a PDGFß-receptor targeted rho kinase inhibitor.

Rho kinase activity in hepatic stellate cells (HSCs) is associated with activation, transformation and contraction of these cells, leading to extracellular matrix production and portal hypertension in liver cirrhosis. Inhibition of rho kinase activity can reduce these activities, but may also lead to side effects, for instance systemic hypotension. This can be circumvented by liver-specific delivery of a rho kinase inhibitor to effector cells. Therefore, we targeted the rho kinase inhibitor Y27632 to the key pathogenic cells in liver fibrosis, i.e. myofibroblasts including activated HSCs that highly express the PDGFß-receptor, using the drug carrier pPB-MSA. This carrier consists of mouse serum albumin (MSA) covalently coupled to several PDGFßR-recognizing moieties (pPB). We aimed to create a prolonged release system of such a targeted construct, by encapsulating pPB-MSA-Y27632 in biodegradable polymeric microspheres, thereby reducing short-lasting peak concentrations and the need for frequent administrations. Firstly, we confirmed the vasodilating potency of PDGFß-receptor targeted Y27632 in vitro in a contraction assay using HSCs seeded on a collagen gel. We subsequently demonstrated the in vivo antifibrotic efficacy of pPB-MSA-Y27632-loaded microspheres in the Mdr2-/- mouse model of progressive biliary liver fibrosis. A single subcutaneous microsphere administration followed by organ harvest one week later clearly attenuated liver fibrosis progression and significantly suppressed the expression of fibrosis related genes, such as several collagens, profibrotic cytokines and matrix metalloproteinases. In conclusion, we demonstrate that polymeric microspheres are suitable as drug delivery system for the sustained systemic delivery of targeted protein constructs with antifibrotic potential, such as pPB-MSA-Y27632. This formulation appears suitable for the sustained treatment of liver fibrosis and possibly other chronic diseases.

Pharmacokinetics of a sustained release formulation of PDGFß-receptor directed carrier proteins to target the fibrotic liver.

Liver fibrogenesis is associated with excessive production of extracellular matrix by myofibroblasts that often leads to cirrhosis and consequently liver dysfunction and death. Novel protein-based antifibrotic drugs show high specificity and efficacy, but their use in the treatment of fibrosis causes a high burden for patients, since repetitive and long-term parenteral administration is required as most proteins and peptides are rapidly cleared from the circulation. Therefore, we developed biodegradable polymeric microspheres for the sustained release of proteinaceous drugs. We encapsulated the drug carrier pPB-HSA, which specifically binds to the PDGFßR that is highly upregulated on activated myofibroblasts, into microspheres composed of hydrophilic multi-block copolymers composed of poly(l-lactide) and poly ethylene glycol/poly(ϵ-caprolactone), allowing diffusion-controlled release. Firstly, we estimated in mice with acute fibrogenesis induced by a single CCl4 injection the half-life of I125-labeled pPB-HSA at 40 min and confirmed the preferential accumulation in fibrotic tissue. Subsequently, we determined in the Mdr2 −/− mouse model of advanced biliary liver fibrosis how the subcutaneously injected microspheres released pPB-HSA into both plasma and fibrotic liver at 24 h after injection, which was maintained for six days. Although the microspheres still contained protein at day seven, pPB-HSA plasma and liver concentrations were decreased. This reduction was associated with an antibody response against the human albumin-based carrier protein, which was prevented by using a mouse albumin-based equivalent (pPB-MSA). In conclusion, this study shows that our polymeric microspheres are suitable as sustained release formulation for targeted protein constructs such as pPB-HSA. These formulations could be applied for the long-term treatment of chronic diseases such as liver fibrosis.

Monitoring biodegradation of diesel fuel in bioventing processes using in situ respiration rate.

An in situ measuring system of respiration rate was applied for monitoring biodegradation of diesel fuel in a bioventing process for bioremediation of diesel contaminated soil. Two laboratory-scale soil columns were packed with 5 kg of soil that was artificially contaminated by diesel fuel as final TPH (total petroleum hydrocarbon) concentration of 8,000 mg/kg soil. Nutrient was added to make a relative concentration of C:N:P = 100:10:1. One soil column was operated with continuous venting mode, and the other one with intermittent (6 h venting/6 h rest) venting mode. On-line O2 and CO2 gas measuring system was applied to measure O2 utilisation and CO2 production during biodegradation of diesel for 5 months. Biodegradation rate of TPH was calculated from respiration rate measured by the on-line gas measuring system. There were no apparent differences between calculated biodegradation rates from two columns with different venting modes. The variation of biodegradation rates corresponded well with trend of the remaining TPH concentrations comparing other biodegradation indicators, such as C17/pristane and C18/phytane ratio, dehydrogenase activity, and the ratio of hydrocarbon utilising bacteria to total heterotrophic bacteria. These results suggested that the on-line measuring system of respiration rate would be applied to monitoring biodegradation rate and to determine the potential applicability of bioventing process for bioremediation of oil contaminated soil.

Role of Ca2+ influx in the tert-butyl hydroperoxide-induced apoptosis of HepG2 human hepatoblastoma cells.

Oxidative stress appears to be implicated in the pathogenesis of various diseases including alcoholic liver injury. In this study we investigated the mechanism of apoptosis induced by tert-butyl hydroperoxide (TBHP) in HepG2 human hepatoblastoma cells. Treatment with TBHP significantly reduced glutathione content and glutathione reductase activity, and increased glutathione peroxidase activity, indicating that TBHP induced oxidative stress in the HepG2 cells. TBHP also induced reduction of cell viability and DNA fragmentation, a hallmark of apoptosis, in a dose-dependent manner. In addition, TBHP induced a sustained increase in intracellular Ca2+ concentration, which was completely prevented by the extracellular Ca2+ chelation with EGTA. TBHP also induced Mn2+ influx. These results indicate that the intracellular Ca2+ increase by TBHP is exclusively due to Ca2+ influx from the extracellular site. Treatment with either an extracellular (EGTA) or an intracellular Ca2+ chelator (BAPTA/AM) significantly suppressed the TBHP-induced apoptosis. Taken together, these results suggest that TBHP induced the apoptotic cell death in the HepG2 cells and that Ca2+ influx may play an important role in the apoptosis induced by TBHP.

Cloning of the thermostable phytase gene (phy) from Bacillus sp. DS11 and its overexpression in Escherichia coli.

Phytase hydrolyzes phytate to release inorganic phosphate, which would decrease the addition of phosphorus to feedstuffs for monogastric animals and thus reduce environmental pollution. The gene encoding phytase from Bacillus sp. DS11 was cloned in Escherichia coli and its sequence determined. A 560-bp DNA fragment was used as a probe to screen the genomic library. It was obtained through PCR of Bacillus sp. DS11 chromosomal DNA and two oligonucleotide primers based on N-terminal amino acid sequences of the purified protein and the cyanogen bromide-cleaved 21-kDa fragment. The phy cloned was encoded by a 2.2-kb fragment. This gene comprises 1152 nucleotides and encodes a polypeptide of 383 amino acids with a deduced molecular mass of 41,808 Da. Phytase was produced to 20% content of total soluble proteins in E. coli BL21 (DE3) using the pET22b(+) vector with the inducible T7 promoter. This is the first nucleic sequence report on phytase from a bacterial strain.

Determination of melatonin in biological samples by capillary electrophoresis.

The determination of melatonin (MLT) in physiological samples was investigated using capillary electrophoresis (CE). Mouse blood was collected in tubes containing EDTA, centrifuged at 1500 g for 20 min at 4 degrees C, and stored at -20 degrees C. Plasma samples were extracted with dichloroethane, centrifuged and the aqueous phase was discarded. Then the organic phase was evaporated to dryness. The residue was dissolved in deionized water and filtered with a microfilter (0.22 micron). Separations were carried out using a CE system equipped with a fused silica capillary [80 cm (effective length 52 cm) x 75 microns I.D.] and an ultraviolet-visible detector (200 nm), and programmed to provide 25 mM 2-(N-morpholino)ethanesulfonic acid (pH 5.7). Injection was performed hydrostatically by elevating the sample by 10 cm at the cathodic side of the capillary. The calibration curve, reproducibility, recovery and limit of detection were examined, and validation of the method was performed. The result showed that MLT in blood could be easily determined with the new method.

Reverse temporalis muscle flap: treatment of large anterior cranial base defect with direct intracranial-nasopharyngeal communication.

Inadequately managed frontal sinus fracture or sinusitis can pose a major problem of intracranial-nasopharyngeal communication. Life-threatening ascending infection of the intracranium is inevitable unless the intracranium is separated from the nasopharynx. In five patients with frontal bone defect associated with direct intracranial-nasopharyngeal communication, the authors used reverse temporalis muscle flap based on the superficial temporal vessels to obliterate the nasocranial communication. With the authors' method, the nasocranial communication were sealed off permanently, and the ascending infection from intracranial-nasopharyngeal communication was controlled successfully. The reverse temporalis muscle flap, which survives by reversed arterial flow through the vascular connection, exists between the superficial temporal artery and the deep temporal artery in the region of temporalis muscle origin. The reverse temporalis muscle flap is versatile and is recommended especially when other local flaps are not available to obliterate the nasocranial communication.