Thermally reversible hydration of beta-chitin

Thermally induced transition between anhydrous and hydrated forms of highly crystalline beta-chitin was studied by differential thermal calorimetry (DSC) and X-ray diffraction. DSC of wet beta-chitin in a sealed pan gave two well-defined endothermic peaks at 85.2 and 104.7 degrees C on heating and one broad exothermic peak at between 60 and 0 degrees C on cooling. These peaks were highly reproducible and became more distinct after repeated heating-cooling cycles. The X-ray diffraction pattern of wet beta-chitin at elevated temperature showed corresponding changes in d-spacing between the sheets formed by stacking of chitin molecules. These phenomena clearly show that water is reversibly incorporated into the beta-chitin crystal and that the temperature change induces transitions between anhydrous, monohydrate, and dihydrate forms. The DSC behavior in heating-cooling cycles, including reversion between the two endothermic peaks, indicated that the transition between monohydrate and dihydrate was a fast and narrow-temperature process, whereas the one between the anhydrous and the monohydrate form was a slow and wide-temperature process.     

Liquid chromatographic-mass spectrometric determination of haloperidol and its metabolites in human plasma and urine

Haloperidol and its two metabolites, reduced haloperidol and 4-(4-chlorophenyl)-4-hydroxypiperidine (CPHP) in human plasma and urine were analyzed by HPLC-MS using a new polymer column (MSpak GF-310), which enabled direct injection of crude biological samples without pretreatment. Recoveries of haloperidol and reduced haloperidol spiked into plasma were 64.4-76.1% and 46.8-50.2%, respectively; those for urine were 87.3-99.4% and 94.2-98.5%, respectively; those of CPHP for both samples were not less than 92.7%. The regression equations for haloperidol, reduced haloperidol and CPHP showed good linearity in the ranges of 10-800, 15-800 and 400-800 ng/ml, respectively, for both plasma and urine. Their detection limits were 5, 10 and 300 ng/ml, respectively, for both samples. Thus, the present method was sensitive enough for detection and determination of high therapeutic and toxic levels for haloperidol and its metabolites present in biological samples.     

Isolation and characterization of a halotolerant <Emphasis Type="Italic">Bacillus</Emphasis><Emphasis Type="Italic">subtilis</Emphasis> BBK-1 which produces three kinds of lipopeptides: bacillomycin L,plipastatin, and surfactin

Twenty-three halotolerant and biosurfactant producing strains were collected from salty conditions in central Thailand. One of the strains designated BBK-1 produced the biosurfactants with the highest activity. BBK-1 was isolated from fermented foods and was identified as B. subtilis based on its physiological characteristics and 16S rRNA gene sequence. We show that the strain grows in media containing NaCl up to 16% (w/v) and produces biosurfactants in NaCl up to 8%. We found that B. subtilis BBK-1 produces three kinds of surface-active lipopeptides simultaneously. By their respective molecular weights and amino acid compositions, it is indicated that these lipopeptides are bacillomycin L, plipastatin, and surfactin. In order to analyze the production mechanism of lipopeptides further in the strain, a generally important biosynthetic gene encoding 4'-phosphopantetheinyl transferase was cloned and sequenced. The gene existed in a single copy in the genome and the deduced amino acid sequence was almost identical to that of Lpa-14 from B. subtilis strain RB14, which co-produces iturin A and surfactin.     

Change of Cu,Zn-superoxide dismutase activity of guinea pig lung in experimental asthma

Correlation between the level of reactive oxygen species (ROS) generated by airway inflammatory cells and superoxide dismutase (SOD) activity of pulmonary tissue during an asthma attach was investigated in a guinea pig model of allergic asthma. In addition, the influence of SOD inhibition by diethyldithiocarbamate (DDC, Cu-chelating agent) on the airway was investigated in terms of pulmonary function during an asthma attach. Relative to controls, the capacity of bronchoalveolar lavage fluid (BAL) cells to release ROS was significantly increased in guinea pigs sensitized with ovalbumin (OA) as the antigen, and significantly increased in guinea pigs with an asthma attack provoked by the inhalation of OA. SOD activity was increased significantly in the antigen-sensitized group. The asthma provocation group showed a tendency for increase in total SOD activity, compared with the sensitization group, whose increase was dependent on the increase in copper, zinc-SOD (Cu, Zn-SOD) activity. Pretreatment with DDC increased the severity and duration of the asthma attack. These results were indicated that Cu, Zn-SOD was closely involved in the asthma process, particularly in the scavenging of oxygen radicals secreted from BAL cells.     

The XPC-HR23B complex displays high affinity and specificity for damaged DNA in a true-equilibrium fluorescence assay

The XPC-HR23B complex is a prime candidate for the initial damage recognition step during global genome nucleotide excision repair. A specific interaction between the XPC-HR23B complex and various types of damaged DNA substrates has been demonstrated in recent work by electrophoretic mobility shift assays or immunoprecipitation. Although these studies allowed the estimation of relative binding affinities for the different types of lesions, the presence of large amounts of competitor DNA or the need for glutaraldehyde fixation prevented the quantification of equilibrium constants. We have performed a quantitative study on the binding of XPC to damaged DNA using fluorescence anisotropy measurements. The XPC-HR23B complex binds with high affinity (K(D) approximately 1-3 nM) to fluorescent 36 bp DNA fragments containing a single cisplatin 1,3-intrastrand adduct or a six-nucleotide mispaired region. From stoichiometric titration experiments, it is concluded that approximately 70% of the XPC-HR23B preparation is active in DNA binding. Binding experiments employing fluorescent probes with a single defined photoproduct reveal a 30-fold preference of XPC for 6,4-photoproducts as compared to a cyclobutane dimer. Competition experiments with undamaged and damaged plasmid DNA indicate that the XPC-HR23B complex discriminates between damaged and undamaged sites with high specificity. The specificity factor is between 100 and 3000, depending on the number of nonspecific sites considered in the calculations. Upon addition of XPA to the XPC binding reaction mixtures, it was not possible to detect cooperative ternary complex formation on the platinated 36 bp probe.     

A role for DNA polymerase beta in mutagenic UV lesion bypass

We report here that DNA polymerase beta (pol beta), the base excision repair polymerase, is highly expressed in human melanoma tissues, known to be associated with UV radiation exposure. To investigate the potential role of pol beta in UV-induced genetic instability, we analyzed the cellular and molecular effects of excess pol beta. We firstly demonstrated that mammalian cells overexpressing pol beta are resistant and hypermutagenic after UV irradiation and that replicative extracts from these cells are able to catalyze complete translesion replication of a thymine-thymine cyclobutane pyrimidine dimer (CPD). By using in vitro primer extension reactions with purified pol beta, we showed that CPD as well as, to a lesser extent, the thymine-thymine pyrimidine-pyrimidone (6-4) photoproduct, were bypassed. pol beta mostly incorporates the correct dATP opposite the 3'-terminus of both CPD and the (6-4) photoproduct but can also misinsert dCTP at a frequency of 32 and 26%, respectively. In the case of CPD, efficient and error-prone extension of the correct dATP was found. These data support a biological role of pol beta in UV lesion bypass and suggest that deregulated pol beta may enhance UV-induced genetic instability.     

Xeroderma pigmentosum group C protein interacts physically and functionally with thymine DNA glycosylase

The XPC-HR23B complex recognizes various helix-distorting lesions in DNA and initiates global genome nucleotide excision repair. Here we describe a novel functional interaction between XPC-HR23B and thymine DNA glycosylase (TDG), which initiates base excision repair (BER) of G/T mismatches generated by spontaneous deamination of 5-methylcytosine. XPC-HR23B stimulated TDG activity by promoting the release of TDG from abasic sites that result from the excision of mismatched T bases. In the presence of AP endonuclease (APE), XPC-HR23B had an additive effect on the enzymatic turnover of TDG without significantly inhibiting the subsequent action of APE. Our observations suggest that XPC-HR23B may participate in BER of G/T mismatches, thereby contributing to the suppression of spontaneous mutations that may be one of the contributory factors for the promotion of carcinogenesis in xeroderma pigmentosum genetic complementation group C patients.     

High Prevalence of the Liver Fluke Amphimerus sp. in Domestic Cats and Dogs in an Area for Human Amphimeriasis in Ecuador

Background

Amphimerus sp. is a liver fluke which recently has been shown to have a high prevalence of infection among an indigenous group, Chachi, who reside in a tropical rainforest in the northwestern region of Ecuador. Since it is unknown which animals can act as a reservoir and/or definitive hosts for Amphimerus sp. in this endemic area, a study was done to determine the prevalence of infection in domestic cats and dogs. This information is important to understand the epidemiology, life cycle and control of this parasite.

Methodology/Findings

In July 2012, three Chachi communities located on Rio Cayapas, province of Esmeraldas, were surveyed. A total of 89 of the 109 registered households participated in the study. Of the 27 cats and 43 dogs found residing in the communities, stool samples were collected from 14 cats and 31 dogs (total of 45 animals) and examined microscopically for the presence of Amphimerus eggs. The prevalence of infection was 71.4% in cats and 38.7% in dogs, with similar rates of infection in all three communities. Significantly more cats were infected than dogs (p = 0.042).

Conclusions/Significance

The data show a high rate of Amphimerus sp. infection in domestic cats and dogs residing in Chachi communities. It can be concluded that these animals act as definitive and reservoir hosts for this liver fluke and that amphimeriasis is a zoonotic disease. These findings provide important epidemiological data which will aid in the development and implementation of control strategies against the transmission of Amphimerus.     

Four new crystal structures of Tk-subtilisin in unautoprocessed, autoprocessed and mature forms: insight into structural changes during maturation

Subtilisin from the hyperthermophilic archaeon Thermococcus kodakaraensis (Tk-subtilisin) is matured from Pro-Tk-subtilisin upon autoprocessing and degradation of the propeptide. The crystal structures of the autoprocessed and mature forms of Tk-subtilisin were determined at 1.89 A and 1.70 A resolution, respectively. Comparison of these structures with that of unautoprocessed Pro-Tk-subtilisin indicates that the structure of Tk-subtilisin is not seriously changed during maturation. However, one unique Ca(2+)-binding site (Ca-7) is identified in these structures. In addition, the N-terminal region of the mature domain (Gly70-Pro82), which binds tightly to the main body in the unautoprocessed form, is disordered and mostly truncated in the autoprocessed and mature forms, respectively. Interestingly, this site is formed also in the unautoprocessed form when its crystals are soaked with 10 mM CaCl(2), as revealed by the 1.87 A structure. Along with the formation of this site, the N-terminal region (Leu75-Thr80) is disordered, with the scissile peptide bond contacting with the active site. These results indicate that the calcium ion binds weakly to the Ca-7 site in the unautoprocessed form, but is trapped upon autoprocessing. We propose that the Ca-7 site is required to promote the autoprocessing reaction by stabilizing the autoprocessed form, in which the new N terminus of the mature domain is structurally disordered. Furthermore, the crystal structure of the Tk-propeptide:S324A-subtilisin complex, which was formed by the addition of separately expressed proteins, was determined at 1.65 A resolution. This structure is virtually identical with that of the autoprocessed form, indicating that the interaction between the two domains is highly intensive and specific.