Isolation and Characterization of the Bovine Microsatellite Loci

Microsatellite loci were isolated using five repetitive probes for Korean native cattle. Eleven microsatellite loci were developed based on a biotin hybrid capture method, and enrichment of the genomic libraries (AAAT, TG, AG, T, and TGC repeats) was performed using Sau3AI adapters. The isolated markers were tested in two half-sib Korean cattle families and four imported breeds (Angus, Limousine, Holstein, and Shorthorn). Nine informative microsatellite loci were observed, and two microsatellite loci were revealed as monomorphic in Korean cattle. In the imported breeds, however, all of the markers were informative. In total, 213 alleles were obtained at the 11 loci across five breeds, and the average number of alleles found per locus, considering all populations, was 4.26. Heterozygosity was 0.71 (expected) and 0.57 (observed). The range of the polymorphic information content for the markers in all cattle populations was 0.43-0.69. Eleven percent of genetic variation was attributed to differentiation between populations as determined by the mean F (ST) values. The remaining 89% corresponded to differences among individuals. The isolated markers may be used to identify and classify the local breeds on a molecular basis.     

Mixotrophy in the newly described phototrophic dinoflagellate Woloszynskia cincta from western Korean waters: feeding mechanism, prey species and effect of prey concentration

Woloszynskia species are dinoflagellates in the order Suessiales inhabiting marine or freshwater environments; their ecophysiology has not been well investigated, in particular, their trophic modes have yet to be elucidated. Previous studies have reported that all Woloszynskia species are photosynthetic, although their mixotrophic abilities have not been explored. We isolated a dinoflagellate from coastal waters in western Korea and established clonal cultures of this dinoflagellate. On the basis of morphology and analyses of the small/large subunit rRNA gene (GenBank accession number=FR690459), we identified this dinoflagellate as Woloszynskia cincta. We further established that this dinoflagellate is a mixotrophic species. We found that W. cincta fed on algal prey using a peduncle. Among the diverse prey provided, W. cincta ingested those algal species that had equivalent spherical diameters (ESDs) ≤12.6 μm, exceptions being the diatom Skeletonema costatum and the dinoflagellate Prorocentrum minimum. However, W. cincta did not feed on larger algal species that had ESDs≥15 μm. The specific growth rates for W. cincta increased continuously with increasing mean prey concentration before saturating at a concentration of ca. 134 ng C/ml (1,340 cells/ml) when Heterosigma akashiwo was used as food. The maximum specific growth rate (i.e. mixotrophic growth) of W. cincta feeding on H. akashiwo was 0.499 d(-1) at 20 °C under illumination of 20 μE/m(2) /s on a 14:10 h light-dark cycle, whereas its growth rate (i.e. phototrophic growth) under the same light conditions without added prey was 0.040 d(-1). The maximum ingestion and clearance rates of W. cincta feeding on H. akashiwo were 0.49 ng C/grazer/d (4.9 cells/grazer/d) and 1.9 μl/grazer/h, respectively. The calculated grazing coefficients for W. cincta on co-occurring H. akashiwo were up to 1.1 d(-1). The results of the present study suggest that grazing by W. cincta can have a potentially considerable impact on prey algal populations.     

Native and non-native conformation-specific antibodies directed to the loop region of hen egg-white lysozyme

Two types of antibodies were differentiated in conventional guinea pig anti-hen egg-white lysozyme (HEL) antisera. The specificities of both antibodies were directed to the loop I region (mainly directed to Cys64--Cys80 loop) but the antibodies were distinct in respect of reactivities with native HEL. One type of antibody reacted with HEL and loop-peptides of HEL but not with the completely reduced and carboxymethylated form of loop-peptides (native conformation specific antibody: NC-Ab). On the other hand, the other type of antibody did not react with HEL but reacted with loop-peptides and also with the completely reduced and carboxymethylated form of loop-peptides (non-native conformation specific antibody: NNC-Ab). The percentage of NNC-Ab in loop I reactive antibody fraction from pooled guinea pig anti-HEL antisera obtained by two different immunization methods was about 25%. Since the affinities of the NNC-Ab to loop-related peptides were higher by one order of magnitude than those of the NC-Ab to the same peptides, care is necessary in evaluating antigenic determinants in native protein. The immunization of guinea pigs with Ploop I . II [sequence 57-107 (Cys64-Cys80, Cys76-Cys94)] evoked an antibody population having specificity similar to but not identical with that of the NNC-Ab type anti-loop I antibody in conventional anti-HEL antisera.     

Identification of Heat Shock Protein 60 as a Regulator of Neutral Sphingomyelinase 2 and Its Role in Dopamine Uptake

Activation of sphingomyelinase (SMase) by extracellular stimuli is the major pathway for cellular production of ceramide, a bioactive lipid mediator acting through sphingomyelin (SM) hydrolysis. Previously, we reported the existence of six forms of neutral pH–optimum and Mg²⁺-dependent SMase (N-SMase) in the membrane fractions of bovine brain. Here, we focus on N-SMase ε from salt-extracted membranes. After extensive purification by 12,780-fold with a yield of 1.3%, this enzyme was eventually characterized as N-SMase2. The major single band of 60-kDa molecular mass in the active fractions of the final purification step was identified as heat shock protein 60 (Hsp60) by matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis. Proximity ligation assay and immunoprecipitation study showed that Hsp60 interacted with N-SMase2, prompting us to examine the effect of Hsp60 on N-SMase2 and ceramide production. Interestingly, Hsp60 siRNA treatment significantly increased the protein level of N-SMase2 in N-SMase2-overexpressed HEK293 cells. Furthermore, transfection of Hsp60 siRNA into PC12 cells effectively increased both N-SMase activity and ceramide production and increased dopamine re-uptake with paralleled increase. Taken together, these results show that Hsp60 may serve as a negative regulator in N-SMase2-induced dopamine re-uptake by decreasing the protein level of N-SMase2.     

Cloning and characterization of two catA genes in Acinetobacter lwoffii K24.

Two novel type I catechol 1,2-dioxygenases inducible on aniline media were isolated from Acinetobacter lwoffii K24. Although the two purified enzymes, CD I1 and CD I2, had similar intradiol cleavage activities, they showed different substrate specificities for catechol analogs, physicochemical properties, and amino acid sequences. Two catA genes, catA1 and catA2, encoding by CD I1 and CD I2, respectively, were isolated from the A. lwoffii K24 genomic library by using colony hybridization and PCR. Two DNA fragments containing the catA1 and catA2 genes were located on separate regions of the chromosome. They contained open reading frames encoding 33.4- and 30.4-kDa proteins. The amino acid sequences of the two proteins matched well with previously determined sequences. Interestingly, further analysis of the two DNA fragments revealed the locations of the catB and catC genes as well. Moreover, the DNA fragment containing catA1 had a cluster of genes in the order catB1-catC1-catA1 while the catB2-catA2-catC2 arrangement was found in the catA2 DNA fragment. These results may provide an explanation of the different substrate specificities and physicochemical properties of CD I1 and CD I2.     

Catalytic mechanism of rhomboid protease GlpG probed by 3,4-dichloroisocoumarin and diisopropyl fluorophosphonate

Rhomboid proteases have many important biological functions. Unlike soluble serine proteases such as chymotrypsin, the active site of rhomboid protease, which contains a Ser-His catalytic dyad, is submerged in the membrane and surrounded by membrane-spanning helices. Previous crystallographic analyses of GlpG, a bacterial rhomboid protease, and its complex with isocoumarin have provided insights into the mechanism of the membrane protease. Here, we studied the interaction of GlpG with 3,4-dichloroisocoumarin and diisopropyl fluorophosphonate, both mechanism-based inhibitors for the serine protease, and describe the crystal structure of the covalent adduct between GlpG and diisopropyl fluorophosphonate, which mimics the oxyanion-containing tetrahedral intermediate of the hydrolytic reaction. The crystal structure confirms that the oxyanion is stabilized by the main chain amide of Ser-201 and by the side chains of His-150 and Asn-154. The phosphorylation of the catalytic Ser-201 weakens its interaction with His-254, causing the catalytic histidine to rotate away from the serine. The rotation of His-254 is accompanied by further rearrangement of the side chains of Tyr-205 and Trp-236 within the substrate-binding groove. The formation of the tetrahedral adduct is also accompanied by opening of the L5 cap and movement of transmembrane helix S5 toward S6 in a direction different from that predicted by the lateral gating model. Combining the new structural data with those on the isocoumarin complex sheds further light on the plasticity of the active site of rhomboid membrane protease.     

Production and characterization of ligninolytic enzymes ofBjerkandera adusta grown on wood meal/wheat bran culture and production of these enzymes using a rotary-solid fermenter

Manganese peroxidase (MnP) and lignin peroxidase (LiP) were produced by growing a white-rot fungusBjerkandera adusta statically, on a wood meal/wheat bran culture in flasks. MnP and LiP reached their maximum activity after 6 and 19 days of inoculation, respectively. Both MnP and LiP are thought to be important enzymes in lignin biodegradation byB. adusta. Ion exchange chromatography showed thatB. adusta produced a single LiP and a single MnP enzyme in wood meal/wheat bran culture. These enzymes were separated and characterized. The molecular weight of MnP was 46,500 with a pl of 3.9. The molecular weight of LiP was estimated to be 47,000 with a pl of 3.5. Spectral analysis demonstrated that both enzymes are heme proteins. Production of these enzymes was also achieved using a rotarysolid culture fermenter. MnP, LiP and veratryl alcohol oxidase were produced byB. adusta in the fermenter.     

RNase III controls the degradation of corA mRNA in Escherichia coli

In Escherichia coli, the corA gene encodes a transporter that mediates the influx of Co(2+), Mg(2+), and Ni(2+) into the cell. During the course of experiments aimed at identifying RNase III-dependent genes in E. coli, we observed that steady-state levels of corA mRNA as well as the degree of cobalt influx into the cell were dependent on cellular concentrations of RNase III. In addition, changes in corA expression levels by different cellular concentrations of RNase III were closely correlated with degrees of resistance of E. coli cells to Co(2+) and Ni(2+). In vitro and in vivo cleavage analyses of corA mRNA identified RNase III cleavage sites in the 5'-untranslated region of the corA mRNA. The introduction of nucleotide substitutions at the identified RNase III cleavage sites abolished RNase III cleavage activity on corA mRNA and resulted in prolonged half-lives of the mRNA, which demonstrates that RNase III cleavage constitutes a rate-determining step for corA mRNA degradation. These findings reveal an RNase III-mediated regulatory pathway that functions to modulate corA expression and, in turn, the influx of metal ions transported by CorA in E. coli.     

Genome-scale metabolic reconstruction and <Emphasis Type="Italic">in silico</Emphasis> analysis of methylotrophic yeast <Emphasis Type="Italic">Pichia pastoris</Emphasis> for strain improvement

Background  

Pichia pastoris has been recognized as an effective host for recombinant protein production. A number of studies have been reported for improving this expression system. However, its physiology and cellular metabolism still remained largely uncharacterized. Thus, it is highly desirable to establish a systems biotechnological framework, in which a comprehensive in silico model of P. pastoris can be employed together with high throughput experimental data analysis, for better understanding of the methylotrophic yeast's metabolism.     

Design of a PKCδ-specific small peptide as a theragnostic agent for glioblastoma

Glioblastoma is an aggressive malignant brain tumor that starts in the brain or spine and frequently recurs after anticancer treatment. The development of an accurate diagnostic system combined with effective cancer therapy is essential to improve prognosis of glioma patients. Peptides, produced from phage display, are attractive biomolecules for glioma treatment because of their biostability, nontoxicity, and small size. In this study, we employed phage display methodology to screen for peptides that specifically recognize the target PKCδ as a novel biomarker for glioma. The phage library screening yielded four different peptides displayed on phages with a 20- to 200-pM K_d value for the recombinant PKCδ catalytic domain. Among these four phage peptides, we selected one to synthesize and tagged it with fluorescein isothiocyanate (FITC) based on the sequence of the PKCδ-binding phage clone. The synthetic peptide showed a relative binding affinity for antibody and localization in the U373 glioma cell. The kinase activity of PKCδ was inhibited by FITC-labeled peptide with an IC₅₀ of 1.4 μM in vitro. Consequently, the peptide found in this study might be a promising therapeutic agent against malignant brain tumor.