Two species of goby, Boleophthalmus pectinirostris and Scartelaos sp., as the new second intermediate hosts of heterophyid fluke in Korea

A survey was performed in order to determine the infection status of the metacercariae of heterophyid fluke in two goby species, Boleophthalmus pectinirostris and Scartelaos sp., collected from Gangjin-gun, and Shinangun, Sooncheon-shi, Jeollanam-do, Republic of Korea. A total of three metacercariae of Heterophyopsis continua was found in only one B. pectinirostris (10.0%) from Gangjin-gun. Heterophyes nocens metacercariae were detected in 24 B. pectinirostris (96.0%) and 14 Scartelaos sp. (63.6%) from Shinan-gun. Heterophyopsis continua metacercariae were found in 11 B. pectinirostris (44.0%) and 21 Scartelaos sp. (95.5%) from Shinan-gun. Stictodora fuscata metacercariae were detected in 18 B. pectinirostris (72.0%) from Shinan-gun. No metacercariae were detected in 20 B. pectinirostris from Sooncheon-shi. From the above results, this study is the first to prove that B. pectinirostris and Scartelaos sp. serve as the second intermediate hosts of some heterophyid flukes in Korea.     

Polymorphisms of the prion protein gene (PRNP) in Hanwoo (Bos taurus coreanae) and Holstein cattle

Polymorphisms in the prion protein gene (PRNP) in humans and sheep correlate with susceptibility to transmissible spongiform encephalopathies (TSEs). Bovine spongiform encephalopathy (BSE) has been reported in British and Japanese cattle; it has occurred thus far in Holstein cattle. BSE in Hanwoo (Bos taurus coreanae) cattle has not been diagnosed up to now. To characterize the bovine PRNP polymorphisms in Korean cattle, we analyzed the open reading frame (ORF) of PRNP in 120 Hanwoo (beef) cattle and 53 Holstein (dairy) cattle. Three polymorphisms were found, the third position of codon 78 (G-->A), the third position of codon 192 (C-->T), and the deletion of a single octa-repeat. An analysis of codon 78 revealed no difference in the genotype (P = 0.2026) or allele (P = 0.7180) frequencies between Hanwoo and Holstein animals. However, there were significant differences in the genotype (P < 0.0001) and allele (P < 0.0001) frequencies at PRNP codon 192 between Hanwoo and Holstein animals. The rate of Holstein animals with deletion of a single octa-repeat was 91.5% undeleted homozygotes, 8.5% heterozygotes (with R3 deletion), and 0% deleted homozygotes. However, none of the 120 Hanwoo animals had any octa-repeat deletions. The genotype (P < 0.0001) and allele (P < 0.0001) frequencies of a single octa-repeat-deletion were also significantly different between Hanwoo and Holstein animals.     

Catabolic role of a three-component salicylate oxygenase from Sphingomonas yanoikuyae B1 in polycyclic aromatic hydrocarbon degradation

Sphingomonas yanoikuyae B1 possesses several different multicomponent oxygenases involved in metabolizing aromatic compounds. Six different pairs of genes encoding large and small subunits of oxygenase iron-sulfur protein components have previously been identified in a gene cluster involved in the degradation of both monocyclic and polycyclic aromatic hydrocarbons. Insertional inactivation of one of the oxygenase large subunit genes, bphA1c, results in a mutant strain unable to grow on naphthalene, phenanthrene, or salicylate. The knockout mutant accumulates salicylate from naphthalene and 1-hydroxy-2-naphthoic acid from phenanthrene indicating the loss of salicylate oxygenase activity. Complementation experiments verify that the salicylate oxygenase in S. yanoikuyae B1 is a three-component enzyme consisting of an oxygenase encoded by bphA2cA1c, a ferredoxin encoded by the adjacent bphA3, and a ferredoxin reductase encoded by bphA4 located over 25kb away. Expression of bphA3-bphA2c-bphA1c genes in Escherichia coli demonstrated the ability of salicylate oxygenase to convert salicylate to catechol and 3-, 4-, and 5-methylsalicylate to methylcatechols.     

Caspase-8 can be activated by interchain proteolysis without receptor-triggered dimerization during drug-induced apoptosis

Proteases of the caspase family are thought to be activated by proteolytic processing of their inactive zymogens. However, although proteolytic cleavage is sufficient for executioner caspases, a different mechanism has been recently proposed for initiator caspases, such as caspase-8, which are believed to be activated by proximity-induced dimerization. According to this model, dimerization rather than proteolytic processing is considered as the critical event for caspase-8 activation. Such a mechanism would suggest that in the absence of a dimerization platform such as the death-inducing signaling complex, caspase-8 proteolytic cleavage would result in an inactive enzyme. As several studies have described caspase-8 cleavage during mitochondrial apoptosis, we now investigated whether caspase-8 becomes indeed catalytically active in this pathway. Using an in vivo affinity labeling approach, we demonstrate that caspase-8 is activated in etoposide-treated cells in vivo in the absence of the receptor-induced death-inducing signaling complex formation. Furthermore, we show that both caspase-3 and -6 are required for the efficient activation of caspase-8. Our data therefore indicate that interchain cleavage of caspase-8 in the mitochondrial pathway is sufficient to produce an active enzyme even in the absence of receptor-driven procaspase-8 dimerization.     

Structural mechanism of oxidative regulation of the phosphatase Cdc25B via an intramolecular disulfide bond

Cdc25B phosphatase, an important regulator of the cell cycle, forms an intramolecular disulfide bond in response to oxidation leading to reversible inactivation of phosphatase activity. We have obtained a crystallographic time course revealing the structural rearrangements that occur in the P-loop as the enzyme goes from its apo state, through the sulfenic (Cys-SO(-)) intermediate, to the stable disulfide. We have also obtained the structures of the irreversibly oxidized sulfinic (Cys-SO(2)(-)) and sulfonic (Cys-SO(3)(-)) Cdc25B. The active site P-loop is found in three conformations. In the apoenzyme, the P-loop is in the active conformation. In the sulfenic intermediate, the P-loop partially obstructs the active site cysteine, poised to undergo the conformational changes that accompany disulfide bond formation. In the disulfide form, the P-loop is closed over the active site cysteine, resulting in an enzyme that is unable to bind substrate. The structural changes that occur in the sulfenic intermediate of Cdc25B are distinctly different from those seen in protein tyrosine phosphatase 1B where a five-membered sulfenyl amide ring is generated as the stable end product. This work elucidates the mechanism by which chemistry and structure are coupled in the regulation of Cdc25B by reactive oxygen species.     

Thermogelling poly(ethylene oxide-b-propylene oxide-b-ethylene oxide) disulfide multiblock copolymer as a thiol-sensitive degradable polymer

We report a reverse thermogelling poly(ethylene oxide-b-propylene oxide-b-ethylene oxide) disulfide multiblock copolymer as a thiol-sensitive biodegradable polymer. The poly(ethylene oxide-b-propylene oxide-b-ethylene oxide) aqueous solutions studied in this research underwent sol-gel-sol or sol-gel-sol-gel transition depending on the molecular weight and concentration of the polymer, whereas the corresponding disulfide multiblock copolymer aqueous solutions underwent sol-gel transition as the temperature increased in a range of 0-60 degrees C. The hydrophobic dye solubilization and dynamic light scattering of the polymer aqueous solution suggest that the poly(ethylene oxide-b-propylene oxide-b-ethylene oxide)s undergo unimer (3 nm) to micelle (12 nm) transition, whereas the disulfide multiblock copolymers undergo unimer (6 nm) to aggregated polymer (600 nm) transition as the temperature increases. The gel duration increased from 6 h (poly(ethylene oxide-b-propylene oxide-b-ethylene oxide)) to more than 12 days (the corresponding disulfide multiblock copolymer) in phosphate buffer saline, and the gel duration of the latter depended on the glutathione concentration of the medium. The model drug, paclitaxel, was released from the in-situ-formed poly(ethylene oxide-b-propylene oxide-b-ethylene oxide) disulfide multiblock copolymer gel in a glutathione concentration-sensitive manner.     

Click synthesis of estradiol–cyclodextrin conjugates as cell compartment selective estrogens

Cyclodextrin (CD) is a well known drug carrier and excipient for enhancing aqueous solubility. CDs themselves are anticipated to have low membrane permeability because of relatively high hydrophilicity and molecular weight. CD derivatization with 17-beta estradiol (E₂) was explored extensively using a number of different click chemistries and the cell membrane permeability of synthetic CD–E₂ conjugate was explored by cell reporter assays and confocal fluorescence microscopy. In simile with reported dendrimer–E₂ conjugates, CD–E₂ was found to be a stable, extranuclear receptor selective estrogen that penetrated into the cytoplasm.