The effects of gamma irradiation on the survival and development of Clonorchis sinensis metacercariae

The effects of gamma irradiation on the survival and development of C. sinensis metacercariae were studied to evaluate the feasibility of irradiation as a control measure for clonorchiasis. Pseudorasbora parva were collected at an endemic river of clonorchiasis and were used for irradiation of the fluke in three schemes. The first (Scheme 1) was irradiation of the isolated metacercariae from the fish followed by infection to experimental rats. The second (Scheme 2) was irradiation of the fish, and then the metacercariae were isolated and infected to rats. The third (Scheme 3) was irradiation on the rat livers after infection with normal metacercariae. Irradiation doses varied from 5 to 100 Gy for Schemes 1 and 2, and 10 to 25 Gy for Scheme 3. The rats were sacrificed 2 to 6 weeks after infection. In Scheme 1, the metacercariae irradiated at 50 Gy failed to survive in the rats after 2 or 6 weeks. However, 1 to 44% of the metacercariae irradiated at 5-30 Gy survived. The estimated LD50 of Scheme 1 was 16.5 Gy. The flukes irradiated in Scheme 2 survived better than those in Scheme 1. The average worm recovery rate in 50 Gy was 28%(7-39% individually). Increasing the dose up to 100 Gy brought a remarkably low survival rate of an average 1%(0-3% individually). The LD50 of Scheme 2 was 47.5 Gy. Worm recovery rates in the 10 Gy group of Scheme 3 were 21-39%, and those in the 25 Gy group were 2% and 34%. Although the metacercariae were irradiated, all of the recovered worms were morphologically normal.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of soybean oil on the enhanced expression of hirudin gene in Hansenula polymorpha

Summary A heterologous gene from bloodsucking leech for anticoagulant, hirudin, has been expressed in the methylotrophic yeast Hansenula polymorpha. The addition of 1%(v/v) soybean oil to the medium as a stabilizer enhanced the expression of the hirudin gene in H. polymorpha with MOX promoter. The production of hirudin in the medium with soybean oil was 320mg/L in a 5L fermenter.     

Tegumental ultrastructure of juvenile and adult Echinostoma cinetorchis]

The tegumental ultrastructure of juvenile and adult Echinostoma cinetorchis (Trematoda: Echinostomatidae) was observed by scanning electron microscopy. Three-day (juvenile) and 16-day (adult) worms were harvested from rats (Sprague-Dawley) experimentally fed the metacercariae from the laboratory-infected fresh water snail, Hippeutis cantori. The worms were fixed with 2.5% glutaraldehyde, processed routinely, and observed by an ISI Korea DS-130 scanning electron microscope. The 3-day old juvenile worms were elongated and ventrally curved, with their ventral sucker near the anterior two-fifths of the body. The head crown was bearing 37-38 collar spines arranged in a zigzag pattern. The lips of the oral and ventral suckers had 8 and 5 type II sensory papillae respectively, and between the spines, a few type III papillae were observed. Tongue or spade-shape spines were distributed anteriorly to the ventral sucker, whereas peg-like spines were distributed posteriorly and became sparse toward the posterior body. The spines of the dorsal surface were similar to those of the ventral surface. The 16-day old adults were leaf-like, and their oral and ventral suckers were located very closely. Aspinous head crown, oral and ventral suckers had type II and type III sensory papillae, and numerous type I papillae were distributed on the tegument anterior to the ventral sucker. Scale-like spines, with broad base and round tip, were distributed densely on the tegument anterior to the ventral sucker but they became sparse posteriorly. At the dorsal surface, spines were observed at times only at the anterior body. The results showed that the tegument of E. cinetorchis is similar to that of other echinostomes, but differs in the number and arrangement of collar spines, shape and distribution of tegumenal spines, and type and distribution of sensory papillae.     

Design and application of highly responsive fluorescence resonance energy transfer biosensors for detection of sugar in living Saccharomyces cerevisiae cells

A protein sensor with a highly responsive fluorescence resonance energy transfer (FRET) signal for sensing sugars in living Saccharomyces cerevisiae cells was developed by combinatorial engineering of the domain linker and the binding protein moiety. Although FRET sensors based on microbial binding proteins have previously been created for visualizing various sugars in vivo, such sensors are limited due to a weak signal intensity and a narrow dynamic range. In the present study, the length and composition of the linker moiety of a FRET-based sensor consisting of CFP-linker(1)-maltose-binding protein-linker(2)-YFP were redesigned, which resulted in a 10-fold-higher signal intensity. Molecular modeling of the composite linker moieties, including the connecting peptide and terminal regions of the flanking proteins, suggested that an ordered helical structure was preferable for tighter coupling of the conformational change of the binding proteins to the FRET response. When the binding site residue Trp62 of the maltose-binding protein was diversified by saturation mutagenesis, the Leu mutant exhibited an increased binding constant (82 microM) accompanied by further improvement in the signal intensity. Finally, the maltose sensor with optimized linkers was redesigned to create a sugar sensor with a new specificity and a wide dynamic range. When the optimized maltose sensors were employed as in vivo sensors, highly responsive FRET images were generated from real-time analysis of maltose uptake of Saccharomyces cerevisiae (baker's yeast).     

Cyclic ADP ribose-dependent Ca2+ release by group I metabotropic glutamate receptors in acutely dissociated rat hippocampal neurons

Group I metabotropic glutamate receptors (group I mGluRs; mGluR1 and mGluR5) exert diverse effects on neuronal and synaptic functions, many of which are regulated by intracellular Ca²⁺. In this study, we characterized the cellular mechanisms underlying Ca²⁺ mobilization induced by (RS)-3,5-dihydroxyphenylglycine (DHPG; a specific group I mGluR agonist) in the somata of acutely dissociated rat hippocampal neurons using microfluorometry. We found that DHPG activates mGluR5 to mobilize intracellular Ca²⁺ from ryanodine-sensitive stores via cyclic adenosine diphosphate ribose (cADPR), while the PLC/IP₃ signaling pathway was not involved in Ca²⁺ mobilization. The application of glutamate, which depolarized the membrane potential by 28.5±4.9 mV (n = 4), led to transient Ca²⁺ mobilization by mGluR5 and Ca²⁺ influx through L-type Ca²⁺ channels. We found no evidence that mGluR5-mediated Ca²⁺ release and Ca²⁺ influx through L-type Ca²⁺ channels interact to generate supralinear Ca²⁺ transients. Our study provides novel insights into the mechanisms of intracellular Ca²⁺ mobilization by mGluR5 in the somata of hippocampal neurons.     

Klebsiella pneumoniae KE-1 degrades endosulfan without formation of the toxic metabolite,endosulfan sulfate

For bioremediation of toxic endosulfan, endosulfan degradation bacteria, which do not form toxic endosulfan sulfate, were isolated from various soil samples using endosulfan as sole carbon and energy source. Among the 40 isolated bacteria, strain KE-1, which was identified as Klebsiella pneumoniae by physiological and 16S rDNA sequence analysis, showed superior endosulfan degradation activity. Analysis of culture pH, growth, free sulfate and endosulfan and its metabolites demonstrated that KE-1 biologically degrades 8.72 microg endosulfan ml(-1) day(-1) when incubated with 93.9 microg ml(-1) endosulfan for 10 days without formation of toxic endosulfan sulfate. Our results suggest that K. pneumoniae KE-1 degraded endosulfan by a non-oxidative pathway and that strain KE-1 has potential as a biocatalyst for endosulfan bioremediation.     

A huge intraductal papillary mucinous carcinoma of the bile duct treated by right trisectionectomy with caudate lobectomy

Background

Leiomyomas of esophagus, although rare, are the most frequent benign tumors of esophagus. Aim of this study is the presentation of 7 patients with esophageal leiomyomas who underwent surgical treatment during a 9-year period.

Methods

Epidemiological data (sex, age), the presenting symptoms, diagnostic examinations, tumor location, histopathological findings and the safety and efficacy of surgical resection are analyzed and assessed.

Results

5 men and 2 women with mean age of 56.9 years were operated. In 3 cases the tumor was located at the lower esophagus, while in the other 4 cases, the leiomyoma was found at the median third of esophagus. 4 patients had severe symptoms related to the leiomyoma, such as dysphagia and epigastric pain. All patients underwent a right postolateral thoracotomy with enucleation of the lesion. None of them received resection of part of the esophagus. The mean diameter of the resected tumors was 4.3 cm. The dimensions of leiomyomas were immediately associated with the symptoms. In no case was detected malignancy or recurrence. All patients were relieved from their symptoms, while postoperative morbidity and mortality did not occur.

Conclusions

Esophageal leiomyoma is a benign tumor, which causes symptoms only if its size becomes large. Surgical enucleation is considered to be safe and effective, without complications.     

Plasmodium vivax: molecular cloning, expression and characterization of glutathione S-transferase

Malaria parasite glutathione S-transferases (GSTs) are postulated to be essential for parasite survival by protecting the parasite against oxidative stress and buffering the detoxification of heme-binding compounds; therefore, GSTs are considered potential targets for drug development. In this study, we identified a Plasmodium vivax gene encoding GST (PvGST) and characterized the biochemical properties of the recombinant enzyme. The PvGST contained 618 bp that encoded 205 amino acids and shared a significant degree of sequence identity with GSTs from other Plasmodium species. The recombinant homodimeric enzyme had an approximate molecular mass of 50kDa and exhibited GSH-conjugating and GSH-peroxidase activities towards various model substrates. The optimal pH for recombinant PvGST (rPvGST) activity was pH 8.0, and the enzyme was moderately unstable at 37 degrees C. The K(m) values of rPvGST with respect to GSH and CDNB were 0.17+/-0.09 and 2.1+/-0.4mM, respectively. The significant sequence homology and similar biochemical properties of PvGST and Plasmodium falciparum GST (PfGST) indicate that they may have similar molecular structures. This information may be useful for the design of specific inhibitors for plasmodial GSTs as potential antimalarial drugs.     

Substrate Binding Mechanism of a Type I Extradiol Dioxygenase

A meta-cleavage pathway for the aerobic degradation of aromatic hydrocarbons is catalyzed by extradiol dioxygenases via a two-step mechanism: catechol substrate binding and dioxygen incorporation. The binding of substrate triggers the release of water, thereby opening a coordination site for molecular oxygen. The crystal structures of AkbC, a type I extradiol dioxygenase, and the enzyme substrate (3-methylcatechol) complex revealed the substrate binding process of extradiol dioxygenase. AkbC is composed of an N-domain and an active C-domain, which contains iron coordinated by a 2-His-1-carboxylate facial triad motif. The C-domain includes a β-hairpin structure and a C-terminal tail. In substrate-bound AkbC, 3-methylcatechol interacts with the iron via a single hydroxyl group, which represents an intermediate stage in the substrate binding process. Structure-based mutagenesis revealed that the C-terminal tail and β-hairpin form part of the substrate binding pocket that is responsible for substrate specificity by blocking substrate entry. Once a substrate enters the active site, these structural elements also play a role in the correct positioning of the substrate. Based on the results presented here, a putative substrate binding mechanism is proposed.