Characterization and identification of Sox2+ radial glia cells derived from rat embryonic cerebral cortex

During the central nervous system (CNS) development, radial glia cells (RGCs) play at least two essential roles, they contribute to neuronal production and the subsequent guidance of neuronal migration, whereas its precise distribution and contribution to cerebral cortex remains less understood. In this research, we used Vimentin as an astroglial marker and Sox2 as a neural progenitor marker to identify and investigate RGCs in rat cerebral cortex at embryonic day (E) 16.5. We found that the Sox2+ progenitor cells localized in the germinal zone (GZ) of E16.5 cerebral cortex, ~95% Sox2+ cells co-localized with Vimentin+ or Nestin+ radial processes which extended to the pial surface across the cortical plate (CP). In vitro, we obtained RG-like cells from E16.5 cerebral cortex on adherent conditions, these Sox2+ Radial glia (RG)-like cells shared some properties with RGCs in vivo, and these Sox2+ RG-like cells could differentiate into astrocytes, oligodendrocytes and presented the radial glia—neuron lineage differentiation ability. Taken together, we identified and investigated some characterizations and properties of Sox2+ RGCs derived from E16.5 cerebral cortex, we suggested that the embryonic Sox2+ progenitor cells which located in the cortical GZ were mainly composed of Sox2+ RGCs, and the cortex-derived Sox2+ RG-like cells displayed the radial glia—neuron lineage differentiation ability as neuronal progenitors in vitro.     

Identification of novel genes responsible for ethanol and/or thermotolerance by transposon mutagenesis in Saccharomyces cerevisiae

Saccharomyces cerevisiae strains tolerant to ethanol and heat stresses are important for industrial ethanol production. In this study, five strains (Tn 1–5) tolerant to up to 15% ethanol were isolated by screening a transposon-mediated mutant library. Two of them displayed tolerance to heat (42 °C). The determination of transposon insertion sites and Northern blot analysis identified seven putative genes (CMP2, IMD4, SSK2, PPG1, DLD3, PAM1, and MSN2) and revealed simultaneous down-regulations of CMP2 and IMD4, and SSK2 and PPG1, down-regulation of DLD3, and disruptions of the open reading frame of PAM1 and MSN2, indicating that ethanol and/or heat tolerance can be conferred. Knockout mutants of these seven individual genes were ethanol tolerant and three of them (SSK2, PPG1, and PAM1) were tolerant to heat. Such tolerant phenotypes reverted to sensitive phenotypes by the autologous or overexpression of each gene. Five transposon mutants showed higher ethanol production and grew faster than the control strain when cultured in rich media containing 30% glucose and initial 6% ethanol at 30 °C. Of those, two thermotolerant transposon mutants (Tn 2 and Tn 3) exhibited significantly enhanced growth and ethanol production compared to the control at 42 °C. The genes identified in this study may provide a basis for the application in developing industrial yeast strains.     

Impacts of organic and inorganic fertilizers on nitrification in a cold climate soil are linked to the bacterial ammonia oxidizer community

The microbiology underpinning soil nitrogen cycling in northeast China remains poorly understood. These agricultural systems are typified by widely contrasting temperature, ranging from −40 to 38°C. In a long-term site in this region, the impacts of mineral and organic fertilizer amendments on potential nitrification rate (PNR) were determined. PNR was found to be suppressed by long-term mineral fertilizer treatment but enhanced by manure treatment. The abundance and structure of ammonia-oxidizing bacterial (AOB) and archaeal (AOA) communities were assessed using quantitative polymerase chain reaction and denaturing gradient gel electrophoresis techniques. The abundance of AOA was reduced by all fertilizer treatments, while the opposite response was measured for AOB, leading to a six- to 60-fold reduction in AOA/AOB ratio. The community structure of AOA exhibited little variation across fertilization treatments, whereas the structure of the AOB community was highly responsive. PNR was correlated with community structure of AOB rather than that of AOA. Variation in the community structure of AOB was linked to soil pH, total carbon, and nitrogen contents induced by different long-term fertilization regimes. The results suggest that manure amendment establishes conditions which select for an AOB community type which recovers mineral fertilizer-suppressed soil nitrification.     

Replication pauses of the wild-type and mutant mitochondrial DNA polymerase gamma: a simulation study

The activity of polymerase γ is complicated, involving both correct and incorrect DNA polymerization events, exonuclease activity, and the disassociation of the polymerase:DNA complex. Pausing of pol-γ might increase the chance of deletion and depletion of mitochondrial DNA. We have developed a stochastic simulation of pol-γ that models its activities on the level of individual nucleotides for the replication of mtDNA. This method gives us insights into the pausing of two pol-γ variants: the A467T substitution that causes PEO and Alpers syndrome, and the exonuclease deficient pol-γ (exo(-)) in premature aging mouse models. To measure the pausing, we analyzed simulation results for the longest time for the polymerase to move forward one nucleotide along the DNA strand. Our model of the exo(-) polymerase had extremely long pauses, with a 30 to 300-fold increase in the time required for the longest single forward step compared to the wild-type, while the naturally occurring A467T variant showed at most a doubling in the length of the pauses compared to the wild-type. We identified the cause of these differences in the polymerase pausing time to be the number of disassociations occurring in each forward step of the polymerase.     

Myotonic dystrophy protein kinase is critical for nuclear envelope integrity

Myotonic dystrophy 1 (DM1) is a multisystemic disease caused by a triplet nucleotide repeat expansion in the 3' untranslated region of the gene coding for myotonic dystrophy protein kinase (DMPK). DMPK is a nuclear envelope (NE) protein that promotes myogenic gene expression in skeletal myoblasts. Muscular dystrophy research has revealed the NE to be a key determinant of nuclear structure, gene regulation, and muscle function. To investigate the role of DMPK in NE stability, we analyzed DMPK expression in epithelial and myoblast cells. We found that DMPK localizes to the NE and coimmunoprecipitates with Lamin-A/C. Overexpression of DMPK in HeLa cells or C2C12 myoblasts disrupts Lamin-A/C and Lamin-B1 localization and causes nuclear fragmentation. Depletion of DMPK also disrupts NE lamina, showing that DMPK is required for NE stability. Our data demonstrate for the first time that DMPK is a critical component of the NE. These novel findings suggest that reduced DMPK may contribute to NE instability, a common mechanism of skeletal muscle wasting in muscular dystrophies.     

Structure-activity relationships of a snake cathelicidin-related peptide, BF-15

Cathelicidin-BF15 (BF-15) is a 15-mer peptide derived from Cathelicidin-BF (BF-30), which is found in the venom of the snake Bungarus fasciatus and exhibits broad antimicrobial activity. Since BF-15 retains most part of the antimicrobial activity of BF-30 but has significantly reduced haemolytic activity and a much shorter sequence length (and less cost), it is a particularly attractive template around which to design novel antimicrobial peptides. However, the structure–activity relationship of it is still unknown. We designed and synthesized a series of C-terminal amidated analogs of BF-15 based on its amphipathic α-helix structure. And we characterized their antimicrobial potency and haemolytic activity. We identified the amidated BF-15 (analog B1) with potent antimicrobial activity against several antibiotic-resistant bacteria (MICs between 1 and 64 μg/mL, 2–16-folds higher than BF-30) and much lower haemolytic activity. The subsequent circular dichroism study results showed a typical α-helix pattern of analog B1 and the content of the α-helix structure of it increased significantly comparing with BF-30, which indicates the peptide sequence of BF-15 may provide a major contribution to the α-helix content of the whole BF-30 sequence. The peptide induced chaotic membrane morphology and cell debris as determined by electron microscopy. This suggests that the antimicrobial activity of B1 is based on cytoplasmic membrane permeability. Taken together, our results suggested that peptide B1 should be considered as an excellent candidate for developing therapeutic drugs.     

Overexpression of <Emphasis Type="Italic">IrlVHA</Emphasis>-<Emphasis Type="Italic">c</Emphasis>, A Vacuolar-Type H<Superscript>+</Superscript>-ATPase c Subunit Gene from <Emphasis Type="Italic">Iris lactea</Emphasis>, Enhances Salt Tolerance in Tobacco

Vacuolar-type H⁺-ATPase (V-ATPase), a multi-subunit endomembrane proton pump, plays an important role in plant growth and response to environmental stresses. In the present study, transgenic tobacco that overexpressed the V-ATPase c subunit gene from Iris lactea (IrlVHA-c) was used to determine the function of IrlVHA-c. Quantitative PCR analysis showed that IrlVHA-c expression was induced by salt stress in I. lactea roots and leaves. Subcellular localization of green fluorescent protein (GFP) as marker combined with FM4-64 staining showed that the IrlVHA-c-GFP was localized to the endosomal compartment in tobacco cells. Compared with the wild-type, the IrlVHA-c transgenic tobacco plants exhibited greater seed germination rates, root length, fresh weight, and higher relative water content (RWC) of leaves under salt stress. Furthermore, the IrlVHA-c transgenic tobacco leaves have lower stomatal densities and larger stomatal apertures than wild-type. Under salt stress, superoxide dismutase (SOD) activity in the transgenic tobacco was significantly enhanced. Moreover, the level of malondialdehyde (MDA) in the transgenic tobacco was significantly lower than that in wild-type plants under salt stress. Taken together, these results suggested that the IrlVHA-c plays an important role in salt tolerance in transgenic tobacco by influencing stomatal movement and physiological changes.