Cloning and expression analysis of a Parkinson's disease gene, uch-L1, and its promoter in zebrafish

Three genes, alpha-synuclein, parkin, and ubiquitin C-terminal hydrolase L1 (UCH-L1), have been associated with inherited forms of Parkinson's disease (PD), although their in vivo functions have remained largely unknown. To develop an animal model for the molecular study of PD, we cloned zebrafish uch-L1 cDNA and its gene promoter. Sequence analysis revealed that the zebrafish Uch-L1 is highly homologous (79%) to the human UCH-L1, which is a member of the deubiquitinating enzymes. By whole-mount in situ hybridization, we examined the spatiotemporal expression of uch-L1 mRNA in developing zebrafish embryos. The uch-L1 mRNAs are detected in neuronal cells at the first day of embryo development. The expression domain of uch-L1 overlaps with that of tyrosine hydroxylase, a molecular marker for dopaminergic neurons, in the ventral diencephalon, an equivalent structure to the substantia nigra where PD progresses in human. To further analyze the tissue-specific regulation of uch-L1 gene expression, we also tested its gene promoter activity and showed a preferential neuronal expression in transient transgenic zebrafish embryos. These results suggest that uch-L1 may have an important role in the development of neuronal cells in early embryos as well as in the degeneration and disease of neuronal cells in late adult brain.     

CAS agar diffusion assay for the measurement of siderophores in biological fluids

We developed a simple and universal method, by modifying the universal CAS (Chrome azurol S) assay, measuring siderophores in various biological fluids. We named the assay as CAS agar diffusion (CASAD) assay. CAS plate devoid of nutrients was prepared by using Bacto-agar (1.5%, w/v) as a matrix. Holes with 5-mm-diameter were punched on the CAS agar plate. Each hole was added by 35 microl of the test fluids containing Desferal that was twofold serially diluted. After incubating at 37 degrees C or room temperature for 4-8 h, the size of orange haloes formed around the holes was measured. The size of orange haloes correlated well with the concentration of Desferal in all the biological fluids tested in this study. CASAD assay showed consistent results in wide pH range from 5 to 9. Addition of iron to the test fluids containing Desferal decreased the size of orange haloes in a dose-dependent manner, which suggests that the CASAD assay detects only iron non-bound siderophore. These results suggest that CASAD assay would serve as a simple, stable, and highly reproducible test for screening and quantitative siderophore analysis in biological fluids.     

Construction of DNA-shuffled and incrementally truncated libraries by a mutagenic and unidirectional reassembly method: changing from a substrate specificity of phospholipase to that of lipase

A method of mutagenic and unidirectional reassembly (MURA) that can generate libraries of DNA-shuffled and randomly truncated proteins was developed. The method involved fragmenting the template gene(s) randomly by DNase I and reassembling the small fragments with a unidirectional primer by PCR. The MURA products were treated with T4 DNA polymerase and subsequently with a restriction enzyme whose site was located on the region of the MURA primer. The N-terminal-truncated and DNA-shuffled library of a Serratia sp. phospholipase A(1) prepared by this method had an essentially random variation of truncated size and also showed point mutations associated with DNA shuffling. After high-throughput screening on triglyceride-emulsified plates, several mutants exhibiting absolute lipase activity (NPL variants) were obtained. The sequence analysis and the lipase activity assay on the NPL variants revealed that N-terminal truncations at a region beginning with amino acids 61 to 71, together with amino acid substitutions, resulted in the change of substrate specificity from a phospholipase to a lipase. We therefore suggest that the MURA method, which combines incremental truncation with DNA shuffling, can contribute to expanding the searchable sequence space in directed evolution experiments.     

Preparation and cell compatibility of acrylamide-grafted poly(3-hydroxyoctanoate)

Poly(3-hydroxyoctanoate) (PHO) films were treated with plasma of different discharge powers (10-50 W) and then treated with acryl amide solutions in order to prepare films with surfaces that contained different amounts of amide groups. The surfaces were characterized by contact angle measurement, attenuated total reflectance infrared spectroscopy, electron spectroscopy for chemical analysis, and scanning electron microscopy. Results from all these measurements indicated that amide groups were present on the surfaces. The amount of amide groups increased in proportion to the discharge power of the plasma. The interaction of Chinese hamster ovary cells with these grafted surfaces was investigated. The number of cells that adhered to and grew on the surfaces was highest for films grafted at 30 W of plasma discharge power, indicating that the moderate hydrophilicity was optimal for cells to adhere and grow. The present results support the suggestion that acryl amide-grafted PHO could be used as cell-compatible biomedical applications.     

Naturally occurring mutations in the largest extracellular loops of ABCA1 can disrupt its direct interaction with apolipoprotein A-I

The ABCA1 transporter contains two large domains into which many of the genetic mutations in individuals with Tangier disease fall. To investigate the structural requirements for the cellular cholesterol efflux mediated by ABCA1, we have determined the topology of these two domains and generated transporters harboring five naturally occurring missense mutations in them. These mutants, unlike wild type ABCA1, produced little or no apoA-I-stimulated cholesterol efflux when transfected into 293 cells, establishing their causality in Tangier disease. Because all five mutant proteins were well expressed and detectable on the plasma membrane, their interaction with the ABCA1 ligand, apolipoprotein (apo) A-I, was measured using bifunctional cross-linking agents. Four of five mutants had a marked decline in cross-linking to apoA-I, whereas one (W590S) retained full cross-linking activity. Cross-linking of apoA-I was temperature-dependent, rapid in onset, and detectable with both lipid- and water-soluble cross-linking agents. These results suggest that apoA-I-stimulated cholesterol efflux cannot occur without a direct interaction between the apoprotein and critical residues in two extracellular loops of ABCA1. The behavior of the W590S mutant indicates that although binding of apoA-I by ABCA1 may be necessary, it is not sufficient for stimulation of cholesterol efflux.     

Degradation of cholesterol by Bacillus subtilis SFF34 isolated from Korean traditional fermented flatfish

AIMS: To examine cholesterol degradation by Bacillus subtilis SFF34. METHODS AND RESULTS: Cholesterol degradation and cholesterol oxidase production by B. subtilis SFF34 were investigated in a medium containing 0.2% cholesterol. In addition, the oxidized product of cholesterol by the purified cholesterol oxidase was detected using a gas chromatograph. Cholesterol oxidase production reached its maximal level (3.14 U ml(-1) after 24 h of incubation in the cholesterol medium. The residual cholesterol content reduced to 0.98 mg g(-1) after 60 h of cultivation in the cholesterol medium. Two cholesterol oxidases were purified from the culture supernatant fluid and their reaction product against cholesterol was identified as 4-cholesten-3-one. CONCLUSIONS: B. subtilis SFF34 degraded cholesterol and produced a high level of extracellular cholesterol oxidase. SIGNIFICANCE AND IMPACT OF THE STUDY: Bacillus subtilis will be very useful for the reduction of cholesterol in many fermented foods and as a source of cholesterol oxidase.     

Differential control of vesicle priming and short-term plasticity by Munc13 isoforms

Presynaptic short-term plasticity is an important adaptive mechanism regulating synaptic transmitter release at varying action potential frequencies. However, the underlying molecular mechanisms are unknown. We examined genetically defined and functionally unique axonal subpopulations of synapses in excitatory hippocampal neurons that utilize either Munc13-1 or Munc13-2 as synaptic vesicle priming factor. In contrast to Munc13-1-dependent synapses, Munc13-2-driven synapses show pronounced and transient augmentation of synaptic amplitudes following high-frequency stimulation. This augmentation is caused by a Ca(2+)-dependent increase in release probability and releasable vesicle pool size, and requires phospholipase C activity. Thus, differential expression of Munc13 isoforms at individual synapses represents a general mechanism that controls short-term plasticity and contributes to the heterogeneity of synaptic information coding.     

Proinflammatory cytokine profile in Vibrio vulnificus septicemic patients' sera

Vibrio vulnificus causes a fulminant and frequently fatal septicemia in susceptible hosts. The present study was designed to evaluate the proinflammatory cytokine profile in V. vulnificus septicemia patients' sera and the effect of doxycycline therapy on the levels of proinflammatory cytokines. Levels of proinflammatory cytokines, tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta and IL-6, were measured in the sera of V. vulnificus septicemic patients and normal healthy volunteers using colorimetric sandwich ELISA. The mean values of TNF-alpha, IL-1beta and IL-6 in the sera of V. vulnificus patients (n=33) increased by 210-, 232- and 40-fold in comparison with those of normal healthy volunteers (n=5), but only the IL-6 level showed a statistically significant difference (P<0.05) between the two groups. Sera from the cases for which doxycycline treatment histories were obvious were designated 'before-treatment' (TX). All the others were included in the after-TX group. In the before-TX group (n=5), the levels of TNF-alpha and IL-1beta significantly increased (P<0.05) in comparison with the after-TX group (n=5). IL-6 levels in the two groups showed no difference. In conclusion, the levels of the well known proinflammatory cytokines TNF-alpha, IL-1beta and IL-6 increased in the V. vulnificus septicemic patients' sera, and the levels of TNF-alpha and IL-1beta decreased significantly after doxycycline treatment. These data indicate that proinflammatory cytokines might play a critical role in V. vulnificus septicemia like in other endotoxemic shocks. The use of doxycycline as an effective bactericidal agent and as an effective modulator of proinflammatory cytokines is supported.