Cloning and expression analysis of Phytoplasma protein translocation genes

Genes encoding SecA and SecY proteins, essential components of the Sec protein translocation system, were cloned from onion yellows phytoplasma, an unculturable plant pathogenic bacterium. The secA gene consists of 2,505 nucleotides encoding an 835 amino acid protein (95.7 kDa) and shows the highest similarity with SecA of Bacillus subtilis. Anti-SecA rabbit antibody was prepared from a purified partial SecA protein, with a histidine tag expressed in Escherichia coli. Western blot analysis confirmed that SecA protein (approximately 96 kDa) is produced in phytoplasma-infected plants. Immunohistochemical thin sections observed by optical microscopy showed that SecA is characteristically present in plant phloem tissues infected with phytoplasma. The secY gene consists of 1,239 nucleotides encoding a 413 amino acid protein (45.9 kDa) and shows the highest similarity with SecY of B. subtilis. These results suggest the presence of a functional Sec system in phytoplasmas. Because phytoplasmas are endocellular bacteria lacking cell walls, this system might secrete bacterial proteins directly into the host cytoplasm. This study is what we believe to be the first report of the sequence and expression analysis of phytoplasma genes encoding membrane proteins with a predicted function.     

Channel Properties of Nax Expressed in Neurons

Na_x is a sodium-concentration ([Na⁺])-sensitive Na channel with a gating threshold of ~150 mM for extracellular [Na⁺] ([Na⁺]_o) in vitro. We previously reported that Na_x was preferentially expressed in the glial cells of sensory circumventricular organs including the subfornical organ, and was involved in [Na⁺] sensing for the control of salt-intake behavior. Although Na_x was also suggested to be expressed in the neurons of some brain regions including the amygdala and cerebral cortex, the channel properties of Na_x have not yet been adequately characterized in neurons. We herein verified that Na_x was expressed in neurons in the lateral amygdala of mice using an antibody that was newly generated against mouse Na_x. To investigate the channel properties of Na_x expressed in neurons, we established an inducible cell line of Na_x using the mouse neuroblastoma cell line, Neuro-2a, which is endogenously devoid of the expression of Na_x. Functional analyses of this cell line revealed that the [Na⁺]-sensitivity of Na_x in neuronal cells was similar to that expressed in glial cells. The cation selectivity sequence of the Na_x channel in cations was revealed to be Na⁺ ≈ Li⁺ > Rb⁺ > Cs⁺ for the first time. Furthermore, we demonstrated that Na_x bound to postsynaptic density protein 95 (PSD95) through its PSD95/Disc-large/ZO-1 (PDZ)-binding motif at the C-terminus in neurons. The interaction between Na_x and PSD95 may be involved in promoting the surface expression of Na_x channels because the depletion of endogenous PSD95 resulted in a decrease in Na_x at the plasma membrane. These results indicated, for the first time, that Na_x functions as a [Na⁺]-sensitive Na channel in neurons as well as in glial cells.     

Scan-Free Absorbance Spectral Imaging A(x,y, λ) of Single Live Algal Cells for Quantifying Absorbance of Cell Suspensions

Label-free, non-invasive, rapid absorbance spectral imaging A(x,y,λ) microscopy of single live cells at 1.2 μm × 1.2 μm resolution with an NA = 0.85 objective was developed and applied to unicellular green algae Chlamydomonas reinhardtii. By introducing the fiber assembly to rearrange a two-dimensional image to the one-dimensional array to fit the slit of an imaging spectrograph equipped with a CCD detector, scan-free acquisition of three-dimensional information of A(x,y,λ) was realized. The space-resolved absorbance spectra of the eyespot, an orange organelle about 1 μm, were extracted from the green-color background in a chlorophyll-rich single live cell absorbance image. Characteristic absorbance change in the cell suspension after hydrogen photoproduction in C. reinhardtii was investigated to find a single 715-nm absorption peak was locally distributed within single cells. The formula to calculate the absorbance of cell suspensions from that of single cells was presented to obtain a quantitative, parameter-free agreement with the experiment. It is quantitatively shown that the average number of chlorophylls per cell is significantly underestimated when it is evaluated from the absorbance of the cell suspensions due to the package effect.     

Shati/Nat8l Overexpression Improves Cognitive Decline by Upregulating Neuronal Trophic Factor in Alzheimer’s Disease Model Mice

Neurochemical Research - Alzheimer’s disease (AD) is a type of dementia characterized by the deposition of amyloid β, a causative protein of AD, in the brain. Shati/Nat8l, identified as...     

Pael-R transgenic mice crossed with parkin deficient mice displayed progressive and selective catecholaminergic neuronal loss

Parkin, a ubiquitin ligase, is responsible for autosomal recessive juvenile parkinsonism (AR-JP). We identified parkin-associated endothelin receptor-like receptor (Pael-R) as a substrate of parkin, whose accumulation is thought to induce unfolded protein response (UPR) -mediated cell death, leading to dopaminergic neurodegeneration. To create an animal model of AR-JP, we generated parkin knockout/Pael-R transgenic (parkin-ko/Pael-R-tg) mice. parkin-ko/Pael-R-tg mice exhibited early and progressive loss of dopaminergic as well as noradrenergic neurons without formation of inclusion bodies, recapitulating the pathological features of AR-JP. Evidence of activation of UPR and up-regulation of dopamine and its metabolites were observed throughout the lifetime. Moreover, complex I activity of mitochondria isolated from parkin-ko/Pael-R-tg mice was significantly reduced later in life. These findings suggest that persistent induction of unfolded protein stress underlies chronic progressive catecholaminergic neuronal death, and that dysfunction of mitochondrial complex I and oxidative stress might be involved in the progression of Parkinson's disease. parkin-ko/Pael-R-tg mice represents an AR-JP mouse model displaying chronic and selective loss of catecholaminergic neurons.     

Genetic mapping of a mutant gene producing three pistils per floret in common wheat

A common wheat (Triticum aestivum L.) mutation that produces 3 pistils (TP) per floret may result in formation of up to 3 kernels per floret. The TP trait may be important for increasing the number of grains per spike and for improving the yield potential through breeding. This trait is determined by the dominant Pis1 gene. Genetic mapping of Pis1 involved 234 microsatellite markers and bulk segregant analysis of a cross of the TP line with Novosibirskaya 67. The Pis1 gene is located on chromosome 2DL, between markers Xgwm539 and Xgwm349. This result does not agree with a previously published localization of the Pis1 gene on chromosome 5B. The possible importance of TP wheat as an alternative genetic resource is discussed.     

Preparation of the Extracellular Domain of Recombinant Human Toll-like Receptor 6

Toll-like receptors (TLRs) mediate immune responses upon recognition of a variety of ligands. To further elucidate the function of TLRs, it is important to identify novel ligands and their action mechanisms including polymer assembly. In this study, we propose an efficient method for preparation of the extracellular domain of human Toll-like receptor 6 (TLR6_ED) in Escherichia coli using the bubbling cultivation method. Our preparation method improved the level of expression of TLR6_ED into a soluble fraction as compared with typical cultivation using a rotary shaker. Circular dichroism (CD) experiments confirmed the structural formation of TLR6_ED with secondary structure contents similar to leucine-rich repeat (LRR) modules. In addition, we also provided a procedure for preparing this recombinant protein using Sf9 insect cells, which ensures preservation of some key posttranslational modifications often lacking in bacteria-expressed proteins. These materials would be useful for analyzing novel molecules that bind directly to TLR6, complex formations with other regulators including TLR2 and TLR4, and the functional effects of N-linked glycosylation.     

A differentially expressed murine RNA encoding a protein with similarities to two types of nucleic acid binding motifs

Using differential screening, a murine cDNA, termed X16, was isolated corresponding to an mRNA which is more strongly expressed in pre-B cell lines relative to mature B-cell lines. The complete coding sequence of the mRNA predicts a 19kD protein with two domains connected by a proline-rich spacer. The N-terminal domain of about 90 amino acids encodes an RNA binding motif including the ribonucleoprotein consensus octapeptide found in one class of RNA-binding proteins and highly conserved from yeast to man. Within the very basic C-terminal domain of about 60 amino acids, several copies of two different peptides are found which are also present in several proteins which bind DNA or RNA. The expression of X16 is not limited to the lymphoid lineage. In adult mice, although the strongest expression was seen in thymus, mRNA was also found in testis, brain, spleen, and very low in heart. X16 mRNA was not detected in liver and kidney. In tissue culture, the expression of X16 mRNA can be induced by serum. The conserved protein motifs and expression pattern suggest that X16 could be involved in RNA processing correlating with cellular proliferation.     

The diversity of interspecific pollen-pistil incongruity in Nicotiana

Nicotiana tabacum was used as a pistillate parent and crossed with three self-compatible species, N. rustica, N. repanda and N. trigonophylla, which were previously reported to have pollen tubes unilaterally inhibited by N. tabacum pistil. Temporal and morphological observations revealed distinct differences of pollen tube behavior among these incongruous crosses. Pollen tubes of N. repanda were arrested in stigma and those of N. rustica in the middle of the style. On the other hand, pollen tubes of N. trigonophylla continued growing at a slow rate. Tubes of N. repanda and N. rustica showed morphological abnormalities such as swelling, thick wall, and irregular callose deposition. In addition, tubes of N. rustica often elongated in reverse direction and wound about in the middle of the style. Although the tubes of N. trigonophylla were apparently normal in morphology, they were distributed throughout the transmitting tissue, differing from the self-pollination of N. tabacum in which they were confined to the peripheral region of it. The diversity of pollen tube behavior indicates that physiological causes of incongruity are different among the three crosses. Bud pollination enabled pollen tubes to reach the ovary in all crosses, indicating that the N. tabacum pistil acquired its ability to inhibit foreign pollen tube elongation with its development. When interspecific hybrids between N. tabacum and the other three species were pollinated by parental species, tubes reached the ovary in all crosses, but the elongation rate of tubes slowed down and morphology was abnormal.     

Effects of fermented green-loofah and green-papaya on nitric oxide secretion from murine macrophage raw 264.7 cells

Molecular Biology Reports - To clarify the effect of lactic acid bacteria (LAB) fermentation on the immunomodulation capacity of green-loofah and green-papaya, aqueous suspensions prepared from the...