Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite morphogenesis at the functional and molecular levels. To aid in these analyses, we have developed a strategy for the isolation of a subclass of PNS neurons called dendritic arborization (da) neurons that have been widely used for studying dendrite morphogenesis^1,2. These neurons are very difficult to isolate as a pure population, due in part to their extremely low occurrence and their difficult-to-reach location below the tough chitinous larval cuticle. Our newly developed method overcomes these challenges, and is based on a fast and specific cell enrichment using antibody-coated magnetic beads. For our magnetic bead sorting studies, we have used age-matched third instar larvae expressing a mouse CD8 tagged GFP fusion protein (UAS-mCD8-GFP)³ under the control of either the class IV dendritic arborization (da) neuron-specific pickpocket (ppk)-GAL4 driver⁴ or the control of the pan-da neuron-specific GAL4^21-7 driver⁵. Although this protocol has been optimized for isolating PNS cells which are attached to the inner wall of the larval cuticle, by varying a few parameters, the same protocol could be used to isolate many different cell types attached to the cuticle at larval or pupal stages of development (e.g. epithelia, muscle, oenocytes etc.), or other cell types from larval organs depending upon the GAL4-specific driver expression pattern. The RNA isolated by this method is of high quality and can be readily used for downstream genomic analyses such as microarray gene expression profiling studies. This approach offers a powerful new tool to perform studies on isolated Drosophila dendritic arborization (da) neurons thereby providing novel insights into the molecular mechanisms underlying dendrite morphogenesis.Download video file.^{(149M, mp4)}     

<Emphasis Type="Italic">Lutibacter oceani</Emphasis> sp. nov., isolated from marine sediment in South Korea

A novel Gram-negative, non-motile, non-spore forming, facultatively anaerobic and short-rod shaped bacterial strain, designated as 325-5^T, was isolated from marine sediment obtained from a coastal region in South Korea. Phylogenetic analysis based on the 16S rRNA gene sequence of strain 325-5^T showed close similarity to Lutibacter crassostreae (97.8 %). The novel isolate was found to grow optimally at 25 °C and pH 7.0. The major fatty acids identified in the strain were iso C_15:0 and iso C_15:0 3OH, which supports the affiliation of strain 325-5^T to the genus Lutibacter. Menaquinone (MK-6) was identified as the respiratory quinone component. The polar lipid profile was found to contain phosphatidylethanolamine and two unidentified lipids. The G+C molar content of strain 325-5^T was determined to be 33.1 mol%. DNA–DNA hybridization of strain 325-5^T exhibited less than 40 % relatedness to L. crassostreae KCTC 42461^T. Pigment analysis showed the presence of carotenoid pigments. Based on this polyphasic analysis, we propose that strain 325-5^T represents a novel species of the genus Lutibacter, for which the name Lutibacter oceani sp. nov. (type strain 325-5^T = JCM 30924^T = KEMB 7306-529^T) is proposed.     

<Emphasis Type="Italic">Tessaracoccus defluvii</Emphasis> sp. nov., isolated from an aeration tank of a sewage treatment plant

A Gram-positive, non-motile, aerobic, coccus-shaped bacterium, designated strain LNB-140^T, was isolated from a sewage treatment plant in the Republic of Korea and was characterised using a polyphasic taxonomic approach. Comparative 16S rRNA gene sequence analysis showed that strain LNB-140^T belongs to genus Tessaracoccus in the family Propionibacteriaceae of the phylum Actinobacteria. The 16S rRNA gene sequence similarities between strain LNB-140^T and type strains of the genus, Tessaracoccus flavescens SST-39^T and Tessaracoccus rhinocerotis YIM101269^T are 97.8 and 97.4 %, respectively. The chemotaxonomic properties of strain LNB-140^T are consistent with those of members of the genus Tessaracoccus: a quinone system with MK-9(H4) as the predominant menaquinone; anteiso-C_15:0 and iso C_15:0 as the predominant cellular fatty acids; and ll-2,6-diaminopimelic acid as the diagnostic peptidoglycan diamino acid. The major polar lipids were identified as diphosphatidylglycerol and phosphatidylethanolamine. The G+C content of the genomic DNA was determined to be 67.1 mol%. Differential phenotypic properties along with low DNA–DNA relatedness (<30 ± 3.2 %) with closely related type strains show that strain LNB-140^T is distinct from previously described members of the genus Tessaracoccus and represents a novel species in this genus, for which the name Tessaracoccus defluvii sp. nov. is proposed. The type strain is LNB-140^T (=KEMB 5401-076^T = JCM 17540^T).     

Effects of temperature on the dynamic behaviour of the HIV-1 nucleocapsid NCp7 and its DNA complex

The nucleocapsid protein NCp7 of human immunodeficiency virus type 1 (HIV-1) contains two highly conserved CCHC zinc fingers and is involved in many crucial steps of the virus life-cycle. A large number of physiological r?les of NCp7 involve its binding to single-stranded nucleic acid chains. Several solution structures of NCp7 and its complex with single-stranded RNA or DNA have been reported. We have investigated the changes in the dynamic behaviour experienced by the (12-53)NCp7 peptide upon DNA binding using (15)N heteronuclear relaxation measurements at 293 K and 308 K, and fluorescence spectroscopy. The relaxation data were interpreted using the reduced spectral density approach, which allowed the high-frequency motion, overall tumbling rates and the conformational exchange contributions to be characterized for various states of the peptide without using a specific motional model. Analysis of the temperature-dependent correlation times derived from both NMR and fluorescence data indicated a co-operative change of the molecular shape of apo (12-53)NCp7 around 303 K, leading to an increased hydrodynamic radius at higher temperatures. The binding of (12-53)NCp7 to a single-stranded d(ACGCC) pentanucleotide DNA led to a reduction of the conformational flexibility that characterized the apo peptide. Translational diffusion experiments as well as rotational correlation times indicated that the (12-53)NCp7/d(ACGCC) complex tumbles as a rigid object. The amplitudes of high-frequency motions were restrained in the complex and the occurrence of conformational exchange was displaced from the second zinc finger to the linker residue Ala30.     

The N-degron approach to create temperature-sensitive mutants in Schizosaccharomyces pombe

Conditional mutants are a vital tool for analysis of gene function. The use of temperature-sensitive mutants in Schizosaccharomyces pombe has significantly promoted understanding of many cellular processes. A portable heat-inducible amino-terminal degron (N-degron) for conditional degradation of a gene product has been previously described in Saccharomyces cerevisiae. This paper describes the adaptation of the N-degron method to create temperature-sensitive (ts) mutants in S. pombe. A ts derivative of the mouse dihydrofolate reductase with an amino-terminal arginine (Arg-DHFR(ts)) previously described in S. cerevisiae was fused to the N-terminus of Bir1p, a nuclear protein involved in mitotic chromosome segregation in S. pombe. This fusion allele, referred to as bir1-td, conferred a chromosome segregation defect at 36 degrees C, as with previously described alleles of bir1. Deletion of the S. pombe E3 ubiquitin ligase (N-recognin), Ubr11p, reversed the temperature-dependent lethality of bir1-td, providing evidence for N-end rule mediated destruction of Bir1p. The methods we describe should therefore facilitate analysis of essential genes in fission yeast for which conditionally lethal mutants are unavailable.     

The S. pombe Cdc14-like phosphatase Clp1p regulates chromosome biorientation and interacts with Aurora kinase

The S. pombe Cdc14-related phosphatase Clp1p/Flp1p regulates G2/M transition by antagonizing CDK activity and is essential for coordinating the nuclear division cycle with cytokinesis through the cytokinesis checkpoint. At the G2/M transition, Clp1p/Flp1p is released from the nucleolus and SPB and distributes throughout the nucleus to the spindle and the contractile ring. This early relocalization is analogous to vertebrate Cdc14 homologs and stands in contrast to S. cerevisiae Cdc14p, which is not released from the nucleolus until metaphase/anaphase transition. Here, we report that Clp1p/Flp1p localizes to kinetochores in prometaphase and functions in chromosome segregation, since deletion of clp1/flp1 causes cosegregation of sister chromatids, when sister kinetochores are prone to mono-orientation. Genetic, cytological, and biochemical experiments suggest that Clp1p/Flp1p functions together with Aurora kinase at kinetochores. Together, these results suggest that Clp1p/Flp1p has a role in repairing mono-orientation of sister kinetochores.     

The Caenorhabditis elegans cell-cycle regulator ZYG-11 defines a conserved family of CUL-2 complex components

The cullin CUL-2 is a crucial component of a subclass of multisubunit cullin-RING ubiquitin-ligases. The specificity of CUL-2-based complexes is provided by variable substrate-recognition subunits that bind to specific substrates. In Caenorhabditis elegans, CUL-2 regulates several key processes in cell division and embryonic development, including meiotic progression, anterior-posterior polarity and mitotic chromatin condensation. However, the substrate recognition subunits that work in these CUL-2-dependent processes were unknown. Here, we present evidence that ZYG-11 is the substrate-recognition subunit for a CUL-2-based complex that regulates these functions. We show that ZYG-11 interacts with CUL-2 in vivo and binds to the complex adaptor protein Elongin C using a nematode variant of the VHL-box motif. We show that the ZYG11 gene family encompasses two main branches in metazoa, and provide evidence that members of the extended ZYG11 family in nematodes and humans are conserved components of CUL2-based ubiquitin-ligases.     

Evidence Suggesting a Role of Iron in a Mouse Model of Nephrogenic Systemic Fibrosis

Nephrogenic systemic fibrosis is associated with gadolinium contrast exposure in patients with reduced kidney function and carries high morbidity and mortality. We have previously demonstrated that gadolinium contrast agents induce in vivo systemic iron mobilization and in vitro differentiation of peripheral blood mononuclear cells into ferroportin (iron exporter)-expressing fibrocytic cells. In the present study we examined the role of iron in a mouse model of nephrogenic systemic fibrosis. Chronic kidney disease was induced in 8-week-old male Balb/C mice with a two-step 5/6 nephrectomy surgery. Five groups of mice were studied: control (n = 5), sham surgery control (n = 5), chronic kidney disease control (n = 4), chronic kidney disease injected with 0.5 mmol/kg body weight of Omniscan 3 days per week, for a total of 10 injections (n = 8), and chronic kidney disease with Omniscan plus deferiprone, 125 mg/kg, in drinking water (n = 9). Deferiprone was continued for 16 weeks until the end of the experiment. Mice with chronic kidney disease injected with Omniscan developed skin changes characteristic of nephrogenic systemic fibrosis including hair loss, reddening, ulceration, and skin tightening by 10 to 16 weeks. Histopathological sections demonstrated dermal fibrosis with increased skin thickness (0.25±0.06 mm, sham; 0.34±+0.3 mm, Omniscan-injected). Additionally, we observed an increase in tissue infiltration of ferroportin-expressing, fibrocyte-like cells accompanied by tissue iron accumulation in the skin of the Omniscan-treated mice. The deferiprone-treated group had significantly decreased skin thickness (p<0.05) and significantly decreased dermal fibrosis compared to the Omniscan-only group. In addition, iron chelation prevented tissue infiltration of ferroportin-expressing, fibrocyte-like cells. Our in vitro experiments demonstrated that exposure to Omniscan resulted in the release of catalytic iron and this was prevented by the iron chelator deferiprone. Deferiprone inhibited the differentiation of human peripheral blood mononuclear cells into ferroportin-expressing cells by immunohistochemical staining and western blot analysis. Our studies support an important role of iron in the pathophysiology of gadolinium chelate toxicity and nephrogenic systemic fibrosis.     

Partial purification and properties of a laundry detergent compatible alkaline protease from a newly isolated <Emphasis Type="Italic">Bacillus</Emphasis> species Y

Alkaline protease production by a newly isolated Bacillus species from laundry soil was studied for detergent biocompatibility. From its morphological and nucleotide sequence (about 1.5 kb) of its 16S rDNA it was identified as Bacillus species with similarity to Bacillus species Y (Gen Bank entry: ABO 55095), and close homology with Bacillus cohnii YN-2000 (Gen Bank entry: ABO23412). Partial purification of the enzyme by ammonium sulfate (50–70% saturation) yielded 8-fold purity. Casein zymography and Sodium dodecylsulphate-Polyacrylamide gel electrophoresis (SDS-PAGE) of the partially purified enzyme revealed two isozymes of molecular sizes approximately 66 kDa and 18 kDa, respectively. The enzyme was most active at pH 12 and 50°C. At pH 12 the enzyme was stable for 5 h and retained 60% activity. The enzyme retained 44% activity at 50°C up to 2 h. The protease showed good hydrolysis specificity with different substrates tested. The presence of Mn²⁺, Co²⁺ and ethylenediaminetetracetic acid (EDTA) showed profound increase in protease activity. The protease of Bacillus species Y showed excellent stability and compatibility with three locally available detergents (Kite, Tide and Aerial) up to 3 h retaining almost 70–80% activity and 10–20% activity at room temperature (30°C) and 50°C, respectively, indicating the potential role of this enzyme for detergent application.