Surface ultrastructure of Metagonimus takahashii metacercariae and adults

A scanning electron microscopic study was performed on the surface ultrastructure of metacercariae and adults of Metagonimus takahashii. Metacercariae were collected from the scale of crucian carp (Carassius auratus), and adult flukes were harvested 1-4 weeks after infection to rats. In excysted metacercariae, the oral sucker had type I (numerous) and type II (seven in total) sensory papillae. Tegumental spines were dense and digitated into 5-7 points on the surface anterior to the ventral sucker, but became sparse and less digitated posteriorly toward the end of the body. In adults, seven type II sensory papillae were characteristically arranged around the lip of the oral sucker, and on the inner side of the lip four small and two large type I sensory papillae were symmetrically seen on each side (12 in total). Tegumental spines on anterior two-thirds of the body, were digitated with 9-12 tips ventrally and 8-13 tips dorsally. Sperms entering into the Laurer's canal were observed. The results show that the surface ultrastructure of M. takahashii is generally similar to those of M. yokogawai and M. miyatai except for the digitation of tegumental spines.     

Persistent endemicity of Gymnophalloides seoi infection in a southwestern coastal village of Korea with special reference to its egg laying capacity in the human host

Follow-up studies have been conducted every three years on the endemicity of Gymnophalloides seoi infection in a small coastal village of Chollanam-do (Province), Korea, since it was first known as an endemic area in 1994. Special attention was given to its egg laying capacity in the human host. In fecal examinations, the overall helminth egg and/or cyst positive rate was 78.7% (74/94) in 1997 and 76.6% (82/107) in 2000. Among them G. seoi eggs showed the highest rate; 71.3% (67/94) in 1997 and 72.0% (77/107) in 2000. The average number of eggs per gram of feces (EPG) was 1,015 in 1997, while a reduced rate of 353 was observed in 2000. In 1997, total of 320,677 adult flukes of G. seoi (av. 10,344/person, 94-69,125 in range) were collected from the diarrheic stools of 31 treated patients. The EPG/worm obtained from 21 cases ranged from 0.04 to 0.77 (av. 0.23), suggesting density-dependent constraints on the worm fecundity. The relationship between the worm burden (X) and EPG/worm (Y) can be expressed as Y = 0.42.e-1.2 chi (r = 0.49). The results showed that G. seoi infection is persistently endemic in this village.     

Toward an understanding of the role of DNA adduct conformation in defining mutagenic mechanism based on studies of the major adduct (formed at N(2)-dG) of the potent environmental carcinogen, benzo[a]pyrene

The process of carcinogenesis is initiated by mutagenesis, which often involves replication past damaged DNA. One question - what exactly is a DNA polymerase seeing when it incorrectly copies a damaged DNA base (e.g., inserting dATP opposite a dG adduct)? - has not been answered in any case. Herein, we reflect on this question, principally by considering the mutagenicity of one activated form of benzo[a]pyrene, (+)-anti-B[a]PDE, and its major adduct [+ta]-B[a]P-N(2)-dG. In previous work, [+ta]-B[a]P-N(2)-dG was shown to be capable of inducing>95% G-->T mutations in one sequence context (5'-TGC), and approximately 95% G-->A mutations in another (5'-AGA). This raises the question - how can a single chemical entity induce different mutations depending upon DNA sequence context? Our current working hypothesis is that adduct conformational complexity causes adduct mutational complexity, where DNA sequence context can affect the former, thereby influencing the latter. Evidence supporting this hypothesis was discussed recently (Seo et al., Mutation Res. [in press]). Assuming this hypothesis is correct (at least in some cases), one goal is to consider what these mutagenic conformations might be. Based on molecular modeling studies, 16 possible conformations for [+ta]-B[a]P-N(2)-dG are proposed. A correlation between molecular modeling and mutagenesis work suggests a hypothesis (Hypothesis 3): a base displaced conformation with the dG moiety of the adduct in the major vs. minor groove gives G-->T vs. G-->A mutations, respectively. (Hypothesis 4, which is a generalized version of Hypothesis 3, is also proposed, and can potentially rationalize aspects of both [+ta]-B[a]P-N(2)-dG and AP-site mutagenesis, as well as the so-called "A-rule".) Finally, there is a discussion of how conformational complexity might explain some unusual mutagenesis results that suggest [+ta]-B[a]P-N(2)-dG can become trapped in different conformations, and why we think it makes sense to interpret adduct mutagenesis results by modeling ds-DNA (at least in some cases), even though the mutagenic event must occur at a ss/ds-DNA junction in the presence of a DNA polymerase.     

Characterization of a bifunctional enzyme fusion of trehalose-6-phosphate synthetase and trehalose-6-phosphate phosphatase of Escherichia coli

To test the effect of the physical proximity of two enzymes catalyzing sequential reactions, a bifunctional fusion enzyme, TPSP, was constructed by fusing the Escherichia coli genes for trehalose-6-phosphate (T6P) synthetase (TPS) and trehalose-6-phosphate phosphatase (TPP). TPSP catalyzes the sequential reaction in which T6P is formed and then dephosphorylated, leading to the synthesis of trehalose. The fused chimeric gene was overexpressed in E. coli and purified to near homogeneity; its molecular weight was 88,300, as expected. The K(m) values of the TPSP fusion enzyme for the sequential overall reaction from UDP-glucose and glucose 6-phosphate to trehalose were smaller than those of an equimolar mixture of TPS and TPP (TPS/TPP). However, the k(cat) values of TPSP were similar to those of TPS/TPP, resulting in a 3.5- to 4.0-fold increase in the catalytic efficiency (k(cat)/K(m)). The K(m) and k(cat) values of TPSP and TPP for the phosphatase reaction from T6P to trehalose were quite similar. This suggests that the increased catalytic efficiency results from the proximity of TPS and TPP in the TPSP fusion enzyme. The thermal stability of the TPSP fusion enzyme was quite similar to that of the TPS/TPP mixture, suggesting that the structure of each enzyme moiety in TPSP is unperturbed by intramolecular constraint. These results clearly demonstrate that the bifunctional fusion enzyme TPSP catalyzing sequential reactions has kinetic advantages over a mixture of both enzymes (TPS and TPP). These results are also supported by the in vivo accumulation of up to 0.48 mg of trehalose per g of cells after isopropyl-beta-D-thiogalactopyranoside treatment of cells harboring the construct encoding TPSP.     

In vivo and in vitro studies of Mgs1 suggest a link between genome instability and Okazaki fragment processing

The non-essential MGS1 gene of Saccharomyces cerevisiae is highly conserved in eukaryotes and encodes an enzyme containing both DNA-dependent ATPase and DNA annealing activities. MGS1 appears to function in post-replicational repair processes that contribute to genome stability. In this study, we identified MGS1 as a multicopy suppressor of the temperature-sensitive dna2Δ405N mutation, a DNA2 allele lacking the N-terminal 405 amino acid residues. Mgs1 stimulates the structure-specific nuclease activity of Rad27 (yeast Fen1 or yFen1) in an ATP-dependent manner. ATP binding but not hydrolysis was sufficient for the stimulatory effect of Mgs1, since non-hydrolyzable ATP analogs are as effective as ATP. Suppression of the temperature-sensitive growth defect of dna2Δ405N required the presence of a functional copy of RAD27, indicating that Mgs1 suppressed the dna2Δ405N mutation by increasing the activity of yFen1 (Rad27) in vivo. Our results provide in vivo and in vitro evidence that Mgs1 is involved in Okazaki fragment processing by modulating Fen1 activity. The data presented raise the possibility that the absence of MGS1 may impair the processing of Okazaki fragments, leading to genomic instability.     

Direct block of cloned hKv1.5 channel by cytochalasins, actin-disrupting agents

The action of cytochalasins, actin-disrupting agents on human Kv1.5 channel (hKv1.5) stably expressed in Ltk^– cells was investigated using the whole cell patch-clamp technique. Cytochalasin B inhibited hKv1.5 currents rapidly and reversibly at +60 mV in a concentration-dependent manner with an IC₅₀ of 4.2 µM. Cytochalasin A, which has a structure very similar to cytochalasin B, inhibited hKv1.5 (IC₅₀ of 1.4 µM at +60 mV). Pretreatment with other actin filament disruptors cytochalasin D and cytochalasin J, and an actin filament stabilizing agent phalloidin had no effect on the cytochalasin B-induced inhibition of hKv1.5 currents. Cytochalasin B accelerated the decay rate of inactivation for the hKv1.5 currents. Cytochalasin B-induced inhibition of the hKv1.5 channels was voltage dependent with a steep increase over the voltage range of the channel's opening. However, the inhibition exhibited voltage independence over the voltage range in which channels are fully activated. Cytochalasin B produced no significant effect on the steady-state activation or inactivation curves. The rate constants for association and dissociation of cytochalasin B were 3.7 µM/s and 7.5 s^–1, respectively. Cytochalasin B produced a use-dependent inhibition of hKv1.5 current that was consistent with the slow recovery from inactivation in the presence of the drug. Cytochalasin B (10 µM) also inhibited an ultrarapid delayed rectifier K⁺ current (I_K,ur) in human atrial myocytes. These results indicate that cytochalasin B primarily blocks activated hKv1.5 channels and endogenous I_K,ur in a cytoskeleton-independent manner as an open-channel blocker. voltage-gated K⁺ channel; heart; open channel block     

Batch and fed-batch production of coenzyme Q10 in recombinant Escherichia coli containing the decaprenyl diphosphate synthase gene from Gluconobacter suboxydans

Coenzyme Q₁₀ (CoQ₁₀) is a quinine consisting of ten units of the isoprenoid side-chain. Because it limits the oxidative attack of free radicals to DNA and lipids, CoQ₁₀ has been used as an antioxidant for foods, cosmetics and pharmaceuticals. Decaprenyl diphosphate synthase (DPS) is the key enzyme for synthesis of the decaprenyl tail in CoQ₁₀ with isopentenyl diphosphate. The ddsA gene coding for DPS from Gluconobacter suboxydans was expressed under the control of an Escherichia coli constitutive promoter. Analysis of the cell extract in recombinant E. coli BL21/pACDdsA by high performance liquid chromatography and mass spectrometry showed that CoQ₁₀ rather than endogenous CoQ₈ was biologically synthesized as the major coenzyme Q. Expression of the ddsA gene with low copy number led to the accumulation of CoQ₁₀ to 0.97 mg l^–1 in batch fermentation. A high cell density (103 g l^–1) in fed-batch fermentation of E. coli BL21/pACDdsA increased the CoQ₁₀ concentration to 25.5 mg l ^–1 and its productivity to 0.67 mg l^–1 h^–1, which were 26.0 and 6.9 times higher than the corresponding values for batch fermentation.     

Aging-related Correlation of Insulin-degrading Enzyme with γ-Secretase-generated Products Involving Insulin and Glucose Levels in Transgenic Mice

Insulin-degrading enzyme (IDE) is a 110-kDa thiol zinc-methalloendopeptidase that can cleave small Aβ peptides and the APP intracellular domain (AICD). The aim of this study was to examine aging-related correlation of IDE with γ-secretase-generated products involving insulin and glucose levels in transgenic brains expressing neuron-specific enolase (NSE)-controlled human mutant presenilin-2 (hPS2m). Herein, we concluded that the levels of IDE expression in transgenic brains were decreased relative to those of control mice at 15 months of age. In parallel, inhibition in the IDE expression at this age underlies to the levels-up of Aβ-42, AICD, γ-secretase, and glucose with a level-down of insulin. Thus, IDE expression is critical target for the therapeutic trials.     

Facilitation of expression and purification of an antimicrobial peptide by fusion with baculoviral polyhedrin in Escherichia coli

Several fusion strategies have been developed for the expression and purification of small antimicrobial peptides (AMPs) in recombinant bacterial expression systems. However, some of these efforts have been limited by product toxicity to host cells, product proteolysis, low expression levels, poor recovery yields, and sometimes an absence of posttranslational modifications required for biological activity. For the present work, we investigated the use of the baculoviral polyhedrin (Polh) protein as a novel fusion partner for the production of a model AMP (halocidin 18-amino-acid subunit; Hal18) in Escherichia coli. The useful solubility properties of Polh as a fusion partner facilitated the expression of the Polh-Hal18 fusion protein ( approximately 33.6 kDa) by forming insoluble inclusion bodies in E. coli which could easily be purified by inclusion body isolation and affinity purification using the fused hexahistidine tag. The recombinant Hal18 AMP ( approximately 2 kDa) could then be cleaved with hydroxylamine from the fusion protein and easily recovered by simple dialysis and centrifugation. This was facilitated by the fact that Polh was soluble during the alkaline cleavage reaction but became insoluble during dialysis at a neutral pH. Reverse-phase high-performance liquid chromatography was used to further purify the separated recombinant Hal18, giving a final yield of 30% with >90% purity. Importantly, recombinant and synthetic Hal18 peptides showed nearly identical antimicrobial activities against E. coli and Staphylococcus aureus, which were used as representative gram-negative and gram-positive bacteria, respectively. These results demonstrate that baculoviral Polh can provide an efficient and facile platform for the production or functional study of target AMPs.