A ß-galactosidase-expressing E. coli culture as an alternative test to identify skin sensitizers and non-sensitizers.

Although the Organization for Economic Cooperation and Development (OECD) has adopted several in vitro methods with reasonable predictive capacity, alternative methods for identifying skin sensitizers and non-sensitizers with reliability and simplicity are still required for more efficient and economic prediction. The present study was to design an in vitro system with the use of a ß-galactosidase-expressing E. coli culture for simpler but sufficiently accurate classification of skin sensitizers and non-sensitizers. A LacZ gene-containing E. coli strain that is capable of producing ß-galactosidase enzyme was induced by isopropyl ß-D-1-thiogalactopyranoside with concomitant treatment with test chemicals. After 6-hr incubation, cells were lysed and ß-galactosidase enzyme activity was monitored colorimetrically by using O-nitrophenyl-D-galactopyranoside as a substrate. Following optimization of several experimental conditions, 22 skin sensitizers and 11 non-sensitizers were examined to assess predictive capacity of this method. The results indicated that predictivity was as follows: 90.9% sensitivity, 81.8% specificity, and 87.9% accuracy, when 17.3% of control activity was used as the cut-off value to separate sensitizers from non-sensitizers. Data suggested that the current bacterial system expressing ß-galactosidase may serve as a useful alternative test for classifying skin sensitizers and non-sensitizers, without the utilization of animals or mammalian cell cultures.

Clinical analysis of an ultrasound system in the evaluation of skin cancers: correlation with histology.

BACKGROUND: The assessment of skin cancers in the clinical setting is often difficult, with important features such as depth and width remaining unknown until the biopsy with pathology reports are received. When we remove skin cancers, with those especially involving the face, aesthetics and invasion to surrounding structures such as bone and cartilage are important features for deciding the optimal surgical procedure and future reconstructive options. The aim of the study was to compare the accuracy of the ultrasound system in vivo and to correlate the results with the histopathological tumor thickness measured in skin cancer patients. PATIENTS AND METHODS: From March 2010 to February 2012, we reviewed 40 patients who comprised a total of 49 skin lesions involving the face, neck, and scalp. Each skin lesions were classified by 9 facial aesthetic units. The patient's various skin lesions were scanned using an ultrasound system device (Philips iU22 xMatrix US), with a 5-17-MHz compact linear transducer. Using the ultrasound system, we analyzed the shape, depth, echogenicity, size, invasion skin level, and vascularity of the skin cancer lesions. The results were correlated with the histology, with special note to the depth of involvement. RESULTS: Of the 40 patients recruited, 15 were male and 25 were female, ranging in age from 53 to 92 years (mean ± SD 78.7 ± 13.7 years). Clinically, 49 lesions suspicious of skin cancer were identified and ultrasounds were performed preoperatively. Depth was measured by ultrasound and histology. Mean ultrasound depth of skin lesion was 3.97 ± 3.15 mm (range 0.80-14.00), and it was found to be 4.04 ± 2.92 mm (range 1.00-14.00) based off of histology. There was excellent correlation (interclass correlation coefficient, 0.953) between the depth of the skin lesions measured histologically and by using the ultrasound. CONCLUSION: The ultrasound is not meant to replace histologic evaluations, but it can be used as another diagnostic tool to provide improved preoperative planning. It can be used as a noninvasive, easy, and low-cost screening method for various skin cancers, and provides valuable information such as lesion margins, shape, layers of involvement, and vascularity patterns.

The role of acyl-coenzyme A carboxylase complex in lipstatin biosynthesis of Streptomyces toxytricini.

Streptomyces toxytricini produces lipstatin, a specific inhibitor of pancreatic lipase, which is derived from two fatty acid moieties with eight and 14 carbon atoms. The pccB gene locus in 10.6 kb fragment of S. toxytricini chromosomal DNA contains three genes for acyl-coenzyme A carboxylase (ACCase) complex accA3, pccB, and pccE that are presumed to be involved in secondary metabolism. The pccB gene encoding a beta subunit of ACCase [carboxyltransferase (CT)] was identified upstream of pccE gene for a small protein of epsilon subunit. The accA3 encoding the alpha subunit of ACCase [biotin carboxylase (BC)] was also identified downstream of pccB gene. When the pccB and pccE genes were inactivated by homologous recombination, the lipstatin production was reduced as much as 80%. In contrast, the accumulation of another compound, tetradeca-5.8-dienoic acid (the major lipstatin precursor), was 4.5-fold increased in disruptant compared with wild-type. It implies that PccB of S. toxytricini is involved in the activation of octanoic acid to hexylmalonic acid for lipstatin biosynthesis.

Identification of pre- and pro-haptens with a ß-galactosidase-expressing E. coli culture system for skin sensitization.

Although several in vitro approaches were successful in separating chemicals as skin sensitizers and non-sensitizers, none of the available methods completely mimics the absolute in vivo scenario of skin sensitization. One of the major challenges with currently available systems would be the limited or no metabolic capacity to activate pre- or pro-haptens to reactive metabolites in the system. In the present study, E. coli cells with ß-galactosidase-expressing LacZ gene were combined with either induced rat liver S-9 fractions or microsomal fractions to detect pre- or pro-haptens to cause skin sensitization. Following optimization of some experimental conditions, we examined 20 sensitizers classified as pre- or pro-haptens and 11 non-sensitizers in these E. coli cultures by incubating bacterial cells and test chemicals with and without S-9 or microsomal proteins. After a 6-h incubation in the presence of IPTG, cells were lyzed to determine the suppression of ß-galactosidase enzyme. A cut-off of 17.3% was applied to determine the percent suppression of ß-galactosidase activity by test chemicals to classify skin sensitizers and non-sensitizers. Among chemicals tested, 19 pre- or pro-haptens were categorized as true positives and 8 non-sensitizers were categorized as true negatives. Thereby, the overall sensitivity, specificity and accuracy achieved with microsome-incorporated and S-9 fraction-incorporated group were 95.0%, 72.7% and 87.1% and 80.0%, 81.8% and 80.6%, respectively. The results suggested that the present bacterial system incorporated with the microsomal activation system could be considered as a useful alternative method to classify not only direct-acting sensitizers but also pre- or pro-haptens requiring metabolic activation in vitro.

Expression and characterization of polyketide synthase module involved in the late step of cephabacin biosynthesis from Lysobacter lactamgenus.

The cephabacins produced by Lysobacter lactamgenus are beta-lactam antibiotics composed of a cephem nucleus, an acetate residue, and an oligopeptide side chain. In order to understand the precise implication of the polyketide synthase (PKS) module in the biosynthesis of cephabacin, the genes for its core domains, beta-ketoacyl synthase (KS), acyltransferase (AT), and acyl carrier protein (ACP), were amplified and cloned into the pET-32b(+) expression vector. The sfp gene encoding a protein that can modify apo-ACP to its active holo-form was also amplified. The recombinant KS, AT, apo-ACP, and Sfp overproduced in the form of His6-tagged fusion proteins in E. coli BL21(DE3) were purified by nickel-affinity chromatography. Formation of stable peptidyl-S-KS was observed by in vitro acylation of the KS domain with the substrate [L-Ala-L-Ala-LAla- L-3H-Arg] tetrapeptide-S-N-acetylcysteamine, which is the evidence for the selective recognition of tetrapeptide produced by nonribosomal peptide synthetase (NRPS) in the NRPS/ PKS hybrid. In order to confirm whether malonyl CoA is the extender unit for acetylation of the peptidyl moiety, the AT domain, ACP domain, and Sfp protein were treated with 14C-malonyl-CoA. The results clearly show that the AT domain is able to recognize the extender unit and decarboxylatively acetylated for the elongation of the tetrapeptide. However, the transfer of the activated acetyl group to the ACP domain was not observed, probably attributed to the improper capability of Sfp to activate apo-ACP to the holo-ACP form.

Biosynthesis of 2-amino-3-hydroxycyclopent-2-enone moiety of bafilomycin in Kitasatospora cheerisanensis KCTC2395.

Bafilomycins produced by Kitasatospora cheerisanensis KCTC- 2395 belong to the 16-membered macrolactone family plecomacrolide antibiotics. Bafilomycin B1 contains 2-amino- 3-hydroxycyclopent-2-enone (C5N), a five membered ring, which gets condensed via an amide linkage to bafilomycin polyketide. To study the biosynthetic pathway of C5N during bafilomycin biosynthesis in K. cheerisanensis KCTC2395, we attempted the functional analysis of two putative genes, encoding 5-aminolevulinic acid synthase (ALAS) and acyl- CoA ligase (ACL). The amplified putative genes for ALAS and ACL were cloned into the E. coli expression vector pET- 32a(+) plasmid, following which the soluble recombinant ALAS and ACL proteins were purified through nickel-affinity column chromatography. Through HPLC analysis of the enzyme reaction mixture, we confirmed the products of putative ALAS and ACL reaction as 5-aminolevulinic acid (5-ALA) and 5-ALA-CoA, respectively. The optimal pH for the putative ALAS reaction was 7.5, and for putative ACL reaction was 7.0, as confirmed by the colorimetric assay. Furthermore, pyridoxal 5'-phosphate (PLP) was found to be an essential cofactor in the putative ALAS reaction, and ATP was a cofactor for the putative ACL catalysis. Finally, we also confirmed that the simultaneous treatment of putative ACL and putative ALAS enzymes resulted in the production of C5N compound from 5-ALA.

Seongsanamides A-D: Antiallergic Bicyclic Peptides from Bacillus safensis KCTC 12796BP.

Six seongsanamides were isolated from the culture broth of Bacillus safensis KCTC 12796BP, and their structures were elucidated by spectroscopic data analysis combined with Marfey's method, electronic circular dichroism calculations, and biosynthetic gene cluster analysis. Compounds 1-4 were bicyclic peptides with isodityrosine residues; 5 and 6 were monocyclic peptides. Only the bicyclic seongsanamides inhibited degranulation and LTC4/PGD2 generation in IgE/Ag-stimulated bone marrow-derived mast cells. Oral administration of 1 suppressed mast cell-dependent passive cutaneous anaphylaxis reaction.

Hepatotoxic and immunotoxic effects produced by 1,3-dibromopropane and its conjugation with glutathione in female BALB/c mice.

To determine a possible role of glutathione (GSH) conjugation in 1,3-dibromopropane (1,3-DBP)-induced hepatotoxicity and immunotoxicity, female BALB/c mice were treated orally with 1,3-DBP. Based on the liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS) analyses, two forms of S-bromopropyl GSH were observed at m/z 427.9 and 429.9 in the positive ESI spectrum with a retention time of 5.29 and 5.23 min, respectively. Following single treatment of mice with 150, 300 or 600 mg/kg 1,3-DBP for 12 hr, the amount of S-bromopropyl GSH was detected maximally in liver homogenates at 600 mg/kg 1,3-DBP. Hepatic GSH levels were significantly decreased by treatment with 1,3-DBP. In a time course study, production of S-bromopropyl GSH rose maximally 6 hr after treatment and decreased gradually thereafter. The liver weights were significantly increased by treatment with 600 mg/kg 1,3-DBP. When mice were treated orally with 600 mg/kg 1,3-DBP for 12 hr, the activities of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were increased by 365- and 83-fold. In addition, oral 1,3-DBP significantly suppressed the antibody response to a T-dependent antigen at 600 mg/kg 1,3-DBP. 1,3-DBP elevated hepatic levels of malondialdehyde and suppressed the activities of some hepatic enzymes involved in anti-oxidation. Taken together, the formation of GSH conjugate with 1,3-DBP may deplete cellular GSH and, subsequently, produce hepatotoxicity and immunotoxicity via damage to the cellular anti-oxidative system.

Substrate specificity of nonribosomal peptide synthetase modules responsible for the biosynthesis of the oligopeptide moiety of cephabacin in Lysobacter lactamgenus.

Lysobacter lactamgenus produces cephabacins, a class of beta-lactam antibiotics which have an oligopeptide moiety attached to the cephem ring at the C-3 position. The nonribosomal peptide synthetase (NRPS) system, which comprises four distinct modules, is required for the biosynthesis of this short oligopeptide, when one takes the chemical structure of these antibiotics into consideration. The cpbI gene, which has been identified in a region upstream of the pcbAB gene, encodes the NRPS - polyketide synthase hybrid complex, where NRPS is composed of three modules, while the cpbK gene -- which has been reported as being upstream of cpbI-- comprises a single NRPS module. An in silico protein analysis was able to partially reveal the specificity of each module. The four recombinant adenylation (A) domains from each NRPS module were heterologously expressed in Escherichia coli and purified. Biochemical data from ATP-PPi exchange assays indicated that L-arginine was an effective substrate for the A1 domain, while the A2, A3 and A4 domains activated L-alanine. These findings are in an agreement with the known chemical structure of cephabacins, as well as with the anticipated substrate specificity of the NRPS modules in CpbI and CpbK, which are involved in the assembly of the tetrapeptide at the C-3 position.

Cloning and analysis of a type II polyketide synthase gene cluster from Streptomyces toxytricini NRRL 15,443.

A standard type II polyketide synthase (PKS) gene cluster was isolated while attempting to clone the biosynthetic gene for lipstatin from Streptomyces toxytricini NRRL 15,443. This result was observed using a Southern blot of a PstI-digested S. toxytricini chromosomal DNA library with a 444 bp amplified probe of a ketosynthase (KS) gene fragment. Four open reading frames [thioesterase (TE), beta-ketoacyl systhase (KAS), chain length factor (CLF), and acyl carrier protein (ACP)], were identified through the nucleotide sequence determination and analysis of a 4.5 kb cloned DNA fragment. In order to confirm the involvement of a cloned gene in lipstatin biosynthesis, a gene disruption experiment for the KS gene was performed. However, the resulting gene disruptant did not show any significant difference in lipstatin production when compared to wild-type S. toxytricini. This result suggests that lipstatin may not be synthesized by a type II PKS.

Identification of medicinal mushroom species based on nuclear large subunit rDNA sequences.

The purpose of this study was to develop molecular identification method for medical mushrooms and their preparations based on the nucleotide sequences of nuclear large subunit (LSU) rDNA. Four specimens were collected of each of the three representative medicinal mushrooms used in Korea: Ganoderma lucidum, Coriolus versicolor, and Fomes fomentarius. Fungal material used in these experiments included two different mycelial cultures and two different fruiting bodies from wild or cultivated mushrooms. The genomic DNA of mushrooms were extracted and 3 nuclear LSU rDNA fragments were amplified: set 1 for the 1.1-kb DNA fragment in the upstream region, set 2 for the 1.2-kb fragment in the middle, and set 3 for the 1.3-kb fragment downstream. The amplified gene products of nuclear large subunit rDNA from 3 different mushrooms were cloned into E. coli vector and subjected to nucleotide sequence determination. The sequence thus determined revealed that the gene sequences of the same medicinal mushroom species were more than 99.48% homologous, and the consensus sequences of 3 different medicinal mushrooms were more than 97.80% homologous. Restriction analysis revealed no useful restriction sites for 6-bp recognition enzymes for distinguishing the 3 sequences from one another, but some distinctive restriction patterns were recognized by the 4-bp recognition enzymes AccII and HhaI. This analysis was also confirmed by PCR-RFLP experiments on medicinal mushrooms.

Effect of ethanol extract of dried Chinese yam (Dioscorea batatas) flour containing dioscin on gastrointestinal function in rat model.

In this study, a 40% ethanol extract of Chinese yam flour (Dioscoreae rhizoma), containing 177 +/- 58 microg/mL of dioscin, was tested in order to evaluate its pharmacological effects on the gastrointestinal tracts of Sprague-Dawley rats. Via the ingestion of the Chinese yam extract, the secretion of gastric acid was suppressed in the rats, and gastrointestinal motility increased by as much as 10%. The fecal quantity of rats fed on the Chinese yam extract also increased, by more than 40% as compared with that of the controls. The Chinese yam extract was found not to affect the growth of normal intestinal bacteria. However, a great deal of lactose-fermenting bacteria was observed in the fecal samples of rats fed for 6 weeks on 2% Chinese yam extract. This finding would appear to suggest that Chinese yam extract not only induces an improvement in digestive capability, but also affects the conversion of some intestinal flora to helpful bacteria. Our serochemical analyses indicated that serum glucose, neutral lipid, and total cholesterol levels were reduced to some degree by long-term feeding on Chinese yam extract. This finding bolsters the notion that Chinese yam extract may prove helpful as a digestion-aiding agent for patients suffering from hyperglycemia or hyperlipidemia.

Global proteomic analysis of protein acetylation affecting metabolic regulation in Daphnia pulex.

Daphnia (Daphnia pulex) is a small planktonic crustacean and a key constituent of aquatic ecosystems. It is generally used as a model organism to study environmental toxic problems. In the past decade, genomic and proteomic datasets of Daphnia have been developed. The proteomic dataset allows for the investigation of toxicological effects in the context of "Daphnia proteomics," resulting in greater insights for toxicological research. To exploit Daphnia for ecotoxicological research, information on the post-translational modification (PTM) of proteins is necessary, as this is a critical regulator of biological processes. Acetylation of lysine (Kac) is a reversible and highly regulated PTM that is associated with diverse biological functions. However, a comprehensive description of Kac in Daphnia is not yet available. To understand the cellular distribution of lysine acetylation in Daphnia, we identified 98 acetylation sites in 65 proteins by immunoprecipitation using an anti-acetyllysine antibody and a liquid chromatography system supported by mass spectroscopy. We identified 28 acetylated sites related to metabolic proteins and six acetylated enzymes associated with the TCA cycle in Daphnia. From GO and KEGG enrichment analyses, we showed that Kac in D. pulex is highly enriched in proteins associated with metabolic processes. Our data provide the first global analysis of Kac in D. pulex and is an important resource for the functional analysis of Kac in this organism.

Analysis of a draft genome sequence of Kitasatospora cheerisanensis KCTC 2395 producing bafilomycin antibiotics.

Kitasatospora cheerisanensis KCTC 2395, producing bafilomycin antibiotics belonging to plecomacrolide group, was isolated from a soil sample at Mt. Jiri, Korea. The draft genome sequence contains 8.04 Mb with 73.6% G+C content and 7,810 open reading frames. All the genes for aerial mycelium and spore formations were confirmed in this draft genome. In phylogenetic analysis of MurE proteins (UDP-N-acetylmuramyl-(L)-alanyl-(D)-glutamate:DAP ligase) in a conserved dcw (division of cell wall) locus, MurE proteins of Kitasatospora species were placed in a separate clade between MurEs of Streptomyces species incorporating (LL)-diaminopimelic acid (DAP) and MurEs of Saccharopolyspora erythraea as well as Mycobacterium tuberculosis ligating meso-DAP. From this finding, it was assumed that Kitasatospora MurEs exhibit the substrate specificity for both (LL)-DAP and meso-DAP. The bafilomycin biosynthetic gene cluster was located in the left subtelomeric region. In 71.3 kb-long gene cluster, 17 genes probably involved in the biosynthesis of bafilomycin derivatives were deduced, including 5 polyketide synthase (PKS) genes comprised of 12 PKS modules.

Association of IL-21 cytokine with severity of primary Sjögren syndrome dry eye.

PURPOSE: IL-21 plays an important role in primary Sjögren syndrome (SS) pathogenesis. The purpose of this study was to evaluate IL-21 expression in tears and the conjunctiva and to analyze the impact of IL-21 on primary SS dry eyes. METHODS: Eighty subjects were enrolled in this study: 30 patients with primary SS dry eye (30 eyes); 30 patients with non-SS dry eye (30 eyes), and 20 normal controls. Tear IL-21 levels were measured by flow cytometry, and IL-21 gene expression in the conjunctiva from impression cytology was evaluated by quantitative polymerase chain reaction. Ocular Surface Disease Index, tear film breakup time, Schirmer I test, and ocular surface staining scores were obtained for all patients. RESULTS: Primary SS dry eyes had significantly higher tear IL-21 levels than non-SS dry eyes and normal controls (P < 0.01). In addition, IL-21 gene expression in the conjunctiva was also higher in primary SS dry eyes than in non-SS dry eyes and normal controls (P < 0.01). However, there were no significant differences in IL-21 expression in tears and the conjunctiva between non-SS dry eyes and controls. The tear IL-21 level was significantly correlated with ocular surface stain scores (r = 0.54, P < 0.01) and Schirmer I test values (r = -0.23, P < 0.05) in primary SS dry eyes. CONCLUSIONS: Our findings suggest that severity of primary SS dry eye is associated with IL-21.

Oxidative Damage and Nuclear Factor Erythroid 2-Related Factor 2 Protein Expression in Normal Skin and Keloid Tissue.

BACKGROUND: Reactive oxygen species (ROS) play an important role in the induction of apoptosis under pathological conditions. Recently, a significant increase in ROS production and disrupted apoptosis mechanisms in keloids have been reported. Nuclear factor erythroid 2-related factor 2 (Nrf2) represents one of the most important cellular defense mechanisms against oxidative stress and is implicated in the regulation of apoptosis. Recently, it has been reported that Nrf2 upregulates Bcl-2, an anti-apoptotic protein. OBJECTIVE: To compare Nrf2 protein expression in normal skin tissues to keloid tissues. METHODS: ROS generation in keloid tissues was evaluated with OxyBlot analysis. Western blotting and/or immunohistochemical staining approaches were used to study expression of Nrf2 or Bcl-2 in keloid and normal skin tissues. Cellular fractionation was performed to examine subcellular distribution of Nrf2. Transfection of fibroblasts with Nrf2-specific small interfering RNA (siRNA) was conducted to understand the relationship between Nrf2 expression and apoptosis induction. RESULTS: Protein oxidation, a marker of oxidative stress, is increased in keloid tissues. Western blot analysis clearly showed that Nrf2 and Bcl-2 are downregulated in keloid tissues. Immunohistochemical staining of Nrf2 confirmed the results of the western blot analysis. Transfection of fibroblasts with the Nrf2-specific siRNA results in increased apoptosis and decreased cell viability. CONCLUSION: Collectively, our data indicate that Nrf2 expression is downregulated in keloid tissues, and that Nrf2 is involved in the development of apoptosis in Nrf2 siRNA-transfected fibroblasts. We propose that a defective antioxidant system and apoptotic dysregulation may participate in keloid pathogenesis.

Asiatic acid, a triterpene, induces apoptosis through intracellular Ca2+ release and enhanced expression of p53 in HepG2 human hepatoma cells.

Asiatic acid (AA), a triterpene, decreased viability and induced apoptosis of HepG2 human hepatoma cells in a dose-dependent manner. AA also markedly increased intracellular Ca(2+) level, which was blocked by TMB-8 and dantrolene, intracellular Ca(2+) release blockers, but not by EGTA, an extracellular Ca(2+) chelator. Moreover, AA-induced apoptosis was significantly suppressed by treatment with TMB-8 and dantrolene, suggesting that intracellular Ca(2+) release may play an essential role in the AA-induced apoptosis. In addition, AA profoundly increased protein level of p53, which was also inhibited by BAPTA/AM, an intracellular Ca(2+) chelator, TMB-8 and dantrolene. Treatment with A23187, a Ca(2+) ionophore, or thapsigargin, a Ca(2+)-ATPase inhibitor, alone enhanced p53 nuclear accumulation, indicating that p53 accumulation is dependent on intracellular Ca(2+) increase. Furthermore, the viability of Hep3B, p53-null cells, was much higher than that of HepG2, p53-wild type cells, when treated with AA. Taken together, these results suggest that AA induced apoptosis through increased intracellular Ca(2+), which, in turn, enhanced p53 expression in HepG2 cells. These results further suggest that AA may be a valuable agent for the therapeutic intervention of human hepatomas.

Cloning and expression of the glucose-1-phosphate thymidylyltransferase gene (gerD) from Streptomyces sp. GERI-155.

GERI-155 is a macrolide antibiotic containing two deoxyhexose molecules, that has antimicrobial activity against Gram-positive bacteria. The deoxysugar biosynthetic gene cluster of GERI-155 was cloned from Streptomyces sp., GERI-155. One of the orfs, gerD, appeared to encode glucose-1-phosphate thymidylyltransferase (dTDP-glucose synthase), which converts dTTP and glucose-1-phosphate to dTDP-D-glucose and pyrophosphate. GerD was expressed in E. coli in vector pHJ2 and the expressed protein was purified to apparent homogeneity by ammonium sulfate precipitation and DEAE-Sepharose CL-6B and DEAE-Trisacryl column chromatography. The specific activity of the enzyme increased 16-fold with a recovery of 10%. It migrated as a single band on SDS-PAGE with a molecular mass of 30 kDa. The purified protein had glucose-1-phosphate thymidylyltransferase activity, catalyzing a reversible bimolecular group transfer reaction. In the forward reaction the highest activity was obtained with the combination of dTTP and alpha-D-glucose-1-phosphate, and only 5.5% of that activity was obtained with UTP in place of dTTP. In the opposite direction the purified protein was highly specific for dTDP-D-glucose and pyrophosphate.

The 5-enolpyruvylshikimate-3-phosphate synthase of glyphosate-tolerant soybean expressed in Escherichia coli shows no severe allergenicity.

The recombinant gene was amplified from the chromosomal DNA of genetically-modified (GM) soybeans and identified as epsps encoding 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) which renders glyphosate resistance. The epsps structural gene was introduced in the pET28(a) plasmid for its expression in Escherichia coli BL21(DE3). It was confirmed that the maximal productivity of the EPSPS protein was achieved when cultivating the recombinant strain in a LB broth for 2 h after supplementing 1 mM isopropylbeta-D-thiogalactopyranoside (IPTG) in a 2 h-culture broth. Since the expressed EPSPS protein was found as an insoluble form in the inclusion body, it was extracted by 6 M urea after sonication, and then purified through immobilized nickel-affinity column chromatography to isolate EPSPS having a molecular mass of 57 kDa. When incubated in simulated gastric fluid containing pepsin at pH 1.5, the purified EPSPS protein was completely digested within 1 min. In addition, the passive cutaneous anaphylaxis reaction of the purified EPSPS protein was not observed in the Sprague Dawley rat system that was administered either orally or subcutaneously. Furthermore, treatment of the EPSPS protein to the culture of the sensitized peritoneal mast cells, or unsensitized but antisera-labeled mast cells, showed neither a remarkable change in the histamine release nor a cytokine production, including interleukin-4 (IL-4) and tumor necrosis factor-alpha (TNF-alpha). Thus, it can be concluded that the EPSPS protein in the GM soybean showed no significant allergenicity in the Sprague Dawley rats.

Characteristics of Dermoid Cyst of the Auricle.

BACKGROUND: Dermoid cysts of the auricular area are extremely rare. We report on six cases of auricular dermoid and epidermoid cyst, and differentiate dermoid cyst from epidermal cyst along with a review of the literature. METHODS: Three cases involved a gradually enlarging mass of the superior and anterior aspect of the helix of their ear. Another two cases were located in the posterior aspect of the ear. RESULTS: During the operation, a tumor was found just under the skin, not fixed mastoid or adjacent cartilage. Histologically, all specimens contained desquamated squamous epithelium and keratin in the lumen. However, two cases of posterior masses showed the presence of adnexal structures and three cases did not. CONCLUSION: A key in diagnosis of the dermoid cyst is the presence of adnexal structures. If the wall does not bear adnexal structures, the term epidermoid or keratin cyst is applied. Acquired cysts are most commonly of traumatic origin and result from an implantation or downward displacement of an epidermal fragment. Finally, the congenital epidermoid cyst grew at the upper part of the auricle; however, the dermoid cyst grew at the lower and posterior part of the auricle.