Proteolytic degradation and potential role of onconeural protein cdr2 in neurodegeneration.

Cerebellar degeneration-related protein 2 (cdr2) is expressed in the central nervous system, and its ectopic expression in tumor cells of patients with gynecological malignancies elicits immune responses by cdr2-specific autoantibodies and T lymphocytes, leading to neurological symptoms. However, little is known about the regulation and function of cdr2 in neurodegenerative diseases. Because we found that cdr2 is highly expressed in the midbrain, we investigated the role of cdr2 in experimental models of Parkinson's disease (PD). We found that cdr2 levels were significantly reduced after stereotaxic injection of 1-methyl-4-phenylpyridinium (MPP(+)) into the striatum. cdr2 levels were also decreased in the brains of post-mortem PD patients. Using primary cultures of mesencephalic neurons and MN9D cells, we confirmed that MPP(+) reduces cdr2 in tyrosine hydroxylase-positive dopaminergic neuronal cells. The MPP(+)-induced decrease of cdr2 was primarily caused by calpain- and ubiquitin proteasome system-mediated degradation, and cotreatment with pharmacological inhibitors of these enzymes or overexpression of calcium-binding protein rendered cells less vulnerable to MPP(+)-mediated cytotoxicity. Consequently, overexpression of cdr2 rescued cells from MPP(+)-induced cytotoxicity, whereas knockdown of cdr2 accelerated toxicity. Collectively, our findings provide insights into the novel regulatory mechanism and potentially protective role of onconeural protein during dopaminergic neurodegeneration.

Genetic differences between the wild and hatchery-produced populations of Korean short barbeled grunter (Hapalogenys nitens) determined with microsatellite markers.

Short barbeled grunter, Hapalogenys nitens, is an economically important fishery resource. In Korea, this fish is in the early stage of domestication, and it has been regarded as the candidate marine fish species for prospective aquaculture diversification. This study presents a preliminary investigation of the future viability of sustainable fry production from short barbeled grunter. We used 12 polymorphic nuclear microsatellite DNA loci to analyze the possible genetic variability between the wild and hatchery-produced populations of short barbeled grunter from Korea and identified 91 alleles. Compared to the wild population, significant genetic changes including reduced genetic diversity (average allele number: 7.42 vs 3.75; average expected heterozygosity: 0.713 vs 0.598, Wilcoxon signed-rank test; P < 0.05) and differentiation [overall fixation index (FST) = 0.088, P < 0.01] occurred in the hatchery-produced population, as indicated by the observation of allele richness, unique allele, heterozygosity, FST, and results of molecular analysis of variance. These findings indicate that genetic drift may have promoted the differentiation between these 2 populations, which may have negative effects on sustainable fry production. Therefore, genetic variations of the wild and hatchery-produced populations should be monitored and subjected to control inbreeding through a commercial breeding program. The information presented by this paper would provide a useful genetic basis for future sustainable culturing planning and management of H. nitens.

Comparative genetic diversity of wild and hatchery-produced populations of tongue sole (Cynoglossus semilaevis) using multiplex PCR assays with polymorphic microsatellite markers.

The tongue sole, Cynoglossus semilaevis (Cynoglossidae), is one of the most economically important fishery resources in Korea. This study presents a preliminary investigation of the future viability of the complete aquaculture of tongue sole in Korea. Specifically, possible differences in genetic variability between wild populations of tongue sole from Korea and hatchery-produced populations of tongue sole from China were assessed using multiplex assays with 12 polymorphic nuclear microsatellite DNA loci. High levels of polymorphism were observed between the 2 populations. A total of 135 different alleles were found, varying from 5-15 alleles per locus, with some alleles being unique. These findings indicate a high level of genetic variability in both the wild and hatchery-produced populations. Although a considerable loss of rare alleles was observed in hatchery samples, there were no statistically significant reductions of heterozygosity or allelic diversity in the hatchery population compared to the wild population. Moreover, the inbreeding coefficient was very low (FIS = -0.010-0.052) for both populations. However, significant genetic heterogeneity was found between the 2 populations. These findings indicate that genetic drift has likely promoted differentiation between these 2 populations, and might have negative effects on the reproductive capacity of the stock, because genetic factors are important in the production of high quality seed for complete aquaculture. Therefore, aquaculture management should incorporate basic genetic principles into existing molecular monitoring protocols. The information compiled by this study is anticipated to provide a useful genetic basis for future complete culturing plans and management of C. semilaevis in fisheries.

Effects of serial passage on the characteristics and chondrogenic differentiation of canine umbilical cord matrix derived mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are often known to have a therapeutic potential in the cell-mediated repair for fatal or incurable diseases. In this study, canine umbilical cord MSCs (cUC-MSCs) were isolated from umbilical cord matrix (n = 3) and subjected to proliferative culture for 5 consecutive passages. The cells at each passage were characterized for multipotent MSC properties such as proliferation kinetics, expression patterns of MSC surface markers and self-renewal associated markers, and chondrogenic differentiation. In results, the proliferation of the cells as determined by the cumulative population doubling level was observed at its peak on passage 3 and stopped after passage 5, whereas cell doubling time dramatically increased after passage 4. Expression of MSC surface markers (CD44, CD54, CD61, CD80, CD90 and Flk-1), molecule (HMGA2) and pluripotent markers (sox2, nanog) associated with self-renewal was negatively correlated with the number of passages. However, MSC surface marker (CD105) and pluripotent marker (Oct3/4) decreased with increasing the number of subpassage. cUC-MSCs at passage 1 to 5 underwent chondrogenesis under specific culture conditions, but percentage of chondrogenic differentiation decreased with increasing the number of subpassage. Collectively, the present study suggested that sequential subpassage could affect multipotent properties of cUC-MSCs and needs to be addressed before clinical applications.

Risks related with withholding and resuming anticoagulation in patients with non-variceal upper gastrointestinal bleeding while on warfarin therapy.

BACKGROUND: The use of warfarin is growing for the prevention or treatment of cardiovascular or cerebrovascular diseases. The risk of haemorrhagic side effects is increased in patients taking warfarin. AIMS: To evaluate risks related with withholding and resuming anticoagulation in patients with upper gastrointestinal bleeding (UGIB) while on warfarin therapy and the role of the second-look endoscopic examination (SEE). METHODS: Records of 58 patients with native valvular heart diseases who presented with non-variceal UGIB during chronic anticoagulation with warfarin were retrospectively reviewed. Age- and gender-matched patients with non-variceal UGIB during aspirin therapy because of ischaemic heart disease were recruited as the control group. RESULTS: Development of both recurrent bleeding and thromboembolic events were more frequent in warfarin group than in control group (7.0% vs. 0% with p = 0.03 and 16.7% vs. 2.4% with p < 0.01, respectively). One of four cases of recurrent bleeding in warfarin group was found by SEE performed in an asymptomatic patient. There were six thromboembolic events which occurred on the 21st, 27th, 28th, 31st, 58th and 75th day from the presentation out of 36 patients who ceased anticoagulation. In contrast, only one from 41 in whom aspirin was discontinued experienced myocardial infarction. There was no difference in the failure of endoscopic haemostasis necessitating angiographic embolisation or surgery, hospital stay, the need of transfusion and overall mortality. CONCLUSIONS: Anticoagulation is recommended to be resumed before the 20th day from the cessation to prevent thromboembolic events. A routine SEE before resuming anticoagulation might be helpful to detect asymptomatic recurrent bleeding.

Investigations on laser hard tissue ablation under various environments.

The purpose of this study was to investigate the effect of liquid environments upon laser bone ablation. A long-pulsed Er,Cr:YSGG laser was employed to ablate bovine bone tibia at various radiant exposures under dry, wet (using water or perfluorocarbon) and spray environmental conditions. Energy loss by the application of liquid during laser irradiation was evaluated, and ablation performance for all conditions was quantitatively measured by optical coherence tomography (OCT). Microscope images were also used to estimate thermal side effects in tissue after multiple-pulse ablation. Wet using water and spray conditions equally attenuated the 2.79 microm wavelength laser beam. Higher transmission efficiency was obtained utilizing a layer of perfluorocarbon. Dry ablation exhibited severe carbonization due to excessive heat accumulation. Wet condition using water resulted in similar ablation volume to the dry case without carbonization. The perfluorocarbon layer produced the largest ablation volume but some carbonization due to the poor thermal conductivity. Spray induced clean cutting with slightly reduced efficiency. Liquid-assisted ablation provided significant beneficial effects such as augmented material removal and cooling/cleaning effects during laser osteotomy.

Hard tissue ablation with a spray-assisted mid-IR laser.

The objective of this study was to understand the dominant mechanism(s) for dental enamel ablation with the application of water spray. A free-running Er,Cr:YSGG (yttrium, scandium, gallium, garnet) laser was used to ablate human enamel tissue at various radiant exposures. During dental ablation, distilled water was sprayed on the sample surface, and these results were compared to ablation without a spray (dry ablation). In order to identify dominant ablation mechanisms, transient acoustic waves were compared to ablation thresholds and the volume of material removed. The ablation profile and depth were measured using optical coherence tomography (OCT). Irregular surface modification, charring and peripheral cracks were associated with dry ablation, whereas craters for spray samples were relatively clean without thermal damage. In spite of a 60% higher ablation threshold for spray associated irradiations owing to water absorption, acoustic peak pressures were six times higher and ablation volume was up to a factor of 2 larger compared to dry ablation. The enhanced pressure and ablation performance of the spray-assisted process was the result of rapid water vaporization, material ejection with recoil stress, interstitial water explosion and possibly liquid-jet formation. With water cooling and abrasive/disruptive mechanical effects, the spray ablation can be a safe and efficient modality for dental treatment.

Ca-alginate microspheres encapsulated in chitosan beads.

Chitosan beads (CBs) incorporating Ca-alginate microspheres (CAMs), containing a drug, were prepared as an oral sustained delivery system. Stable and monodisperse Ca-alginate microspheres loaded with drug were obtained by a membrane emulsification method. The Ca-alginate microspheres were encapsulated in chitosan beads by the ionotropic gelation method with a polyelectrolyte complex reaction between two oppositely charged polyions. The surface and internal characteristics of the beads were improved by ionic cross-linking in tripolyphosphate (TPP) solution adjusted to pH 5.0. The release experiments were performed using lidocaine.HCl (cationic drug) and sodium salicylate (anionic drug) as model drugs. Initial release of drugs depended on the degree of swelling. Ca-alginate microspheres encapsulated in chitosan beads were superior to both drug-loaded CBs and CAMs beads for sustained release because they had a three-layer composition; a calcium alginate core bounded by an inter-phasic chitosan-alginate membrane, which itself was surrounded by a layer of chitosan-TPP.

Calculus fragmentation in laser lithotripsy.

The intracorporeal treatment of urinary calculi with lasers is presented, which describes laser-calculus interactions associated with lithotripsy. Reliable fragmentation of calculi with diverse compositions and minimal collateral tissue damage are primarily contingent upon laser parameters (wavelength, pulse duration, and pulse energy) and physical properties of calculi (optical, mechanical, and chemical). The pulse duration governs the dominant mechanism in calculi fragmentation, which is either photothermal or photoacoustical/photomechanical. Lasers with long pulse durations (i.e. > tens of micros) induce a temperature rise in the laser-affected zone with minimal acoustic waves; material is removed by means of vaporization, melting, mechanical stress, and/or chemical decomposition. Short-pulsed laser ablation (i.e. < 10 micros), on the other hand, produces shock waves, and the resultant mechanical energy fragments calculi. Work continues throughout the world to evaluate the feasibility of advanced lasers in lithotripsy and to optimize laser parameters and light delivery systems pertinent to efficient fragmentation of calculi.

Tobacco clones derived from tissue culture with supersensitivity to ozone.

At least two supersensitive tobacco somaclones were obtained from tissue culture (TC) , when this approach was used to asexually propagate Bel-W3 tobacco indicator plants. These somaclones can detect as low as 30 ppb ozone for a 4-h exposure duration both within CSTR exposure chambers and in ambient air. Comparison of the injury index and their coefficient of variance showed that the TC plantlets usually have more uniform performance in response to ozone in addition to their higher sensitivity. A quick regeneration procedure was established to preserve the supersensitive germplasm immediately when it was found. The TC plantlets will flower and produce seed similar to seed-grown tobacco. The TC approach proved to be a better propagation system for valuable indicator plant species. The mechanism that causes the variation and the possible difference in their genome from seed-grown tobacco is still unknown. Further studies are needed in the future to determine if factors in the TC system may be responsible for the sensitivity difference.

Targeting of MPEG-protected polyamino acid carrier to human E-selectin in vitro.

Targeted diagnostic agents are expected to have a significant impact in molecular imaging of cell-surface associated markers of proliferation, inflammation and angiogenesis. In this communication, we describe a new class of targeted polyamino acid-based protected graft copolymers (PGC) of poly-(L-lysine) and methyl poly-(ethylene glycol) (PGC) covalently conjugated with a monoclonal antibody fragment, F(ab')(2). We utilized targeted PGC conjugates as carriers of near-infrared indocyanine fluorophores (Cy5.5) for optical imaging of endothelial cell populations expressing IL-1 beta inducible proinflammatory marker E-selectin. We compared two conjugation chemistries, involving either introduction of sulfhydryl group to F(ab')(2), or via direct attachment of the antibody fragment directly to the chemically activated PGC. Both PGC-based targeted agents demonstrated high binding specificity (20-30 fold over non-specific uptake) and were utilized for imaging E-selectin expression on human endothelial cells activated with IL-1 beta.

Inhibitory action of water soluble fraction of Terminalia chebula on systemic and local anaphylaxis.

We investigated the effects of the water soluble fraction of Terminalia chebula (Combretaceae) (WFTC) on systemic and local anaphylaxis. WFTC administered 1h before compound 48/80 injection inhibited compound 48/80-induced anaphylactic shock 100% with doses of 0.01-1.0 g/kg. When WFTC was administered 5 or 10 min after compound 48/80 injection, the mortality also decreased in a dose-dependent manner. Passive cutaneous anaphylaxis was inhibited by 63.5+/-7.8% by oral administration of WFTC (1.0 g/kg). When WFTC was pretreated at concentrations ranging from 0.005 to 1.0 g/kg, the serum histamine levels were reduced in a dose-dependent manner. WFTC (0.01-1.0 mg/ml) also significantly inhibited histamine release from rat peritoneal mast cells (RPMC) by compound 48/80. However, WFTC (1.0 mg/ml) had a significant increasing effect on anti-dinitrophenyl IgE-induced tumor necrosis factor-alpha production from RPMC. These results indicate that WFTC may possess a strong antianaphylactic action.

Fabrication of porous gelatin scaffolds for tissue engineering.

A novel method which employs water present in swollen hydrogels as a porogen for shape template was suggested for preparing porous materials. Biodegradable hydrogels were prepared through crosslinking of gelatin with glutaraldehyde in aqueous solution, followed by rinsing and washing. After freezing the swollen hydrogels, the ice formed within the hydrogel network was sublimated by freeze-drying. This simple method produced porous hydrogels. Irrespective of any rinsing and washing processes, water was homogeneously distributed into the hydrogel network, allowing the hydrogel network to uniformly enlarge and the ice to act as a porogen during the freezing process. Different porous structures were obtained by varying the freezing temperature. Hydrogels frozen in liquid nitrogen, had a two-dimensionally ordered structure, while the hydrogels prepared at freezing temperatures near -20 degrees C, showed a three-dimensional structure with interconnected pores. As the freezing temperature was lowered, the hydrogel structure gradually became more two-dimensionally ordered. These results suggest that the porosity of dried hydrogels can be controlled by the size of ice crystals formed during freezing. It was concluded that the present freeze-drying procedure is a bio-clean method for formulating biodegradable sponges of different pore structures without use of any additives and organic solvents.

Phylogenetic analysis of Erwinia species based on 16S rRNA gene sequences.

The phylogenetic relationships of the type strains of 16 Erwinia species were investigated by performing a comparative analysis of the sequences of the 16S rRNA genes of these organisms. The sequence data were analyzed by the neighbor-joining method, and each branch was supported by moderate bootstrap values. The phylogenetic tree and sequence analyses confirmed that the genus Erwinia is composed of species that exhibit considerable heterogeneity and form four clades that are intermixed with members of other genera, such as Escherichia coli, Klebsiella pneumoniae, and Serratia marcescens. Cluster I includes the type strains of Erwinia herbicola, Erwinia milletiae, Erwinia ananas, Erwinia uredovora, and Erwinia stewartii and corresponds to Dye's herbicola group. Cluster II consists of Erwinia persicinus, Erwinia rhapontici, Erwinia amylovora, and Erwinia cypripedii. Cluster III consists of Erwinia carotovora subspecies and Erwinia chrysanthemi and is characterized by the production of pectate lyases and cellulases. Erwinia salicis, Erwinia rubrifaciens, and Erwinia nigrifluens form the cluster that is most distantly related to other Erwinia species. The data from the sequence analyses are discussed in the context of biochemical and DNA-DNA hybridization data.

Cellular localization and functional analysis of the protein encoded by the chromosomal virulence gene(acvB) of Agrobacterium tumefaciens.

A chromosomal virulence gene, acvB, of Agrobacterium tumefaciens [J. Bacteriol., 175, 3208-3212 (1993)] was over-expressed in Escherichia coli. A 47-kDa protein was produced and localized in the periplasmic space of E. coli. Amino acid sequence analysis of its N-terminal demonstrated that a signal peptide of 24 amino acids was cleaved from the pre AcvB protein to produce the mature 47-kDa protein. Western-blot analysis using the antiserum against the AcvB protein detected a 47-kDa protein in the periplasmic space only with strain A208 (acvB+). The amount of AcvB protein synthesized was not increased in strain A208 by induction with acetosyringone (100 microM). There was observed no significant difference in induction by acetosyringone of virB::lacZ, virD::lacZ, and virE::lacZ fusion genes regardless of the presence or absence of the acvB gene. The T-strand (lower strand of T-DNA) was detected in strains A208 as well as B119 (acvB-) which were cultured in induction medium containing acetosyringone. AcvB protein bound to single-stranded DNAs with no apparent sequence specificity. The results suggest that AcvB protein binds to the T-strand in periplasm and mediates the transfer of the T-strand from A. tumefaciens to the host plant cell.

Isolation and characterization of a new chromosomal virulence gene of Agrobacterium tumefaciens.

A mutant (strain B119) of Agrobacterium tumefaciens with a chromosomal mutation was isolated by transposon (Tn5) mutagenesis. The mutant exhibited growth rates on L agar and minimal medium (AB) plates similar to those of the parent strain (strain A208 harboring a nopaline-type Ti plasmid). The mutant was avirulent on all host plants tested: Daucus carota, Cucumis sativus, and Kalanchoe diagremontiana. The mutant was not impaired in attachment ability to carrot cells. The mutant had one insertion of Tn5 in its chromosome. The avirulent phenotype of B119 was shown to be due to the Tn5 insertion in the chromosome by the marker exchange technique. A wild-type target chromosomal segment (3.0 kb) which included the site of mutation was cloned and sequenced. Two open reading frames, ORF-1 (468 bp) and ORF-2 (995 bp), were identified in the 3.0-kb DNA segment. Tn5 was inserted in the middle of ORF-2 (acvB gene). Introduction of the acvB gene into the mutant B119 strain complemented the avirulent phenotype of the strain. Homology search found no genes homologous to acvB, although it had some similarity to the open reading frame downstream of the virA gene on the Ti plasmid. Thus, the acvB gene identified in this study seems to be a new chromosomal virulence gene of A. tumefaciens.

Isolation and genetic analysis of an Agrobacterium tumefaciens avirulent mutant with a chromosomal mutation produced by transposon mutagenesis.

A transposon 5 (Tn5) insertion was introduced into the genome of A. tumefaciens (A-208 strain harboring a nopaline type Ti-plasmid) using a conjugative pJB4JI plasmid containing Tn5. Five thousand transconjugants were assayed for virulence on carrot (Daucus carota L.) disks; 54 isolates were avirulent or very attenuated. The cellular localization (plasmid or chromosome) of the Tn5 insertion in those isolates were identified by Southern hybridization analysis. An avirulent mutant (B-90 strain) with the Tn5 insertion in the chromosome was selected and characterized. The mutant had the same growth rate as that of the parent strain in L-broth. The mutant and the parent strain had similar attachment ability to carrot root cells. Tn5 was inserted into one site of the chromosome. The wild-type target chromosomal region (1281 base pairs) was cloned and sequenced. An open reading frame (ORF) consisting of 395 base pairs was identified. The wild-type DNA fragment (1.6 kb) containing the ORF introduced into B-90 strain complemented the avirulent phenotype of the strain. A soluble protein was predicted from the ORF. The Tn5 was inserted near the 3'-terminal of the ORF. Homology search of this ORF found no significant homology to known genes and proteins. Thus, the ORF identified in this paper seems to be a new chromosomal virulence gene of A. tumefaciens.

Pulmonary lymphangioleiomyomatosis in a male.

Pulmonary lymphangioleiomyomatosis has been observed almost exclusively in women, usually in their reproductive years. Exacerbations with pregnancy and after hormonal manipulation have been documented, and it has been suggested that its pathogenesis is due to the influence of hormonal(estrogenic) stimulus. The clinical, roentgenographic, and histopathologic features of this case of pulmonary lymphangioleiomyomatosis in a 22-year-old male are all characteristic of those described in prior reports, except for the patient's sex. With the following case of pulmonary lymphangioleiomyomatosis in a male, we suggest the possibility of the existence of an additional pathogenetic mechanism.