Cloning and truncation modification of trehalose-6-phosphate synthase gene from Selaginella pulvinata

A homologous sequence was amplified from resurrection plant Selaginella pulvinta by RACE technique, proved to be the full-length cDNA of trehalose-6-phosphate synthase gene by homologous alignment and yeast complementation assay, and nominated as SpTPS1 gene. The open reading frame of this gene was truncated 225 bp at the 5′-end, resulting the N-terminal truncation modification of 75 amino acids for its encoding protein. The TPS1 deletion mutant strain YSH290 of the brewer's yeast transformed by the truncated gene SpTPS1Δ and its original full-length version restored growth on the medium with glucose as a sole carbon source and displayed growth curves with no significant difference, indicating their encoding proteins functioning as TPS enzyme. The TPS activity of the mutant strain transformed by the truncated gene SpTPS1Δ was about six fold higher than that transformed by its original version, reasoning that the extra N-terminal extension of the full-length amino acid sequence acts as an inhibitory domain to trehalose synthesis. However, the trehalose accumulation of the mutant strain transformed by the truncated gene SpTPS1Δ was only 8% higher than that transformed by its original version. This result is explained by the feedback balance of trehalose content coordinated by the comparative activities between trehalose synthase and trehalase. The truncated gene SpTPS1Δ is suggested to be used in transgenic operation, together with the inhibition of trehalase activity by the application of validamycin A or genetic deficiency of the endogenous trehalase gene, for the enhancement of trehalose accumulation and improvement of abiotic tolerance in transgenic plants.     

Dynamic response of HPV16/anti-HPV16 pairs with unbinding events studied by atomic force microscopy

This paper proposes an effective approach to distinguish whether samples include Human Papilloma virus type-16 (HPV16) by Atomic force microscopy (AFM). AFM is an important instrument in nanobiotechnology field. At first we identified the HPV16 by Polymerase chain reaction (PCR) analysis and Western blotting from specimen of the HPV patient (E12) and the normal (C2), and then we used an AFM to observe the surface ultrastructure by tapping mode and to measure the unbinding force between HPV16 coupled to an AFM tip and anti-HPV16 L1 coated on the substrate surface by contact mode. The experimental results by tapping mode show that the size of a single HPV viron was similar to its SEM image from the previous literatures; moreover, based on the purposed methods and the analysis, two obvious findings that we can determine whether or not the subject is a HPV patient can be derived from the results; one is based on the distribution of unbinding forces, and the other is based on the distribution of the stiffness. Furthermore, the proposed method could be a useful technique for further investigating the potential role among subtypes of HPVs in the oncogenesis of human cervical cancer.     

Altered plasma acylcarnitine and amino acid profiles in type 2 diabetic kidney disease

Introduction

Dysregulation of acylcarnitines (AcylCNs) and amino acids metabolism have implicated in abnormality of fatty acid oxidation in type 2 diabetes (T2D). However, it is not well known whether altered plasma AcylCN, and amino acid profiles are associated with albuminuria or diabetic nephropathy (DN) in T2D.

Objective

The aim of this study was to elucidate alterations in plasma levels of AcylCNs and amino acids with respect to the T2D patients with various stages of albuminuria.

Methods

We recruited 52 healthy subjects as control, and 156 T2D patients which were divided into 52 normoalbuminuria, 52 microalbuminuria, and 52 macroalbuminuria. Plasma 37 AcylCNs and 12 amino acids were analyzed by tandem mass spectrometry.

Results

We found that T2D with normoalbuminuria and microalbuminuria had lower shot-, medium-, and long-chain AcylCNs, whereas T2D with macroalbuminuria had higher short-and medium-chain AcylCNs and lower long-chain AcylCNs than healthy subjects. Moreover, estimated glomerular filtration rate (eGFR) was a negative, independent and significant predictor of albumin to creatinine ratio (ACR) levels (β = ?0.376, P < 0.001), whereas plasma Low-density lipoprotein cholesterol (LDL-C) was significantly and positively associated with ACR levels (β = 0.169, P = 0.049). Furthermore, multivariate ordinal logistic regression analysis revealed that isobutyrylcarnitine (C4) was a positive, independent, and significant predictor of ACR levels with higher odds of having T2D patients with progression normoalbuminuria to microalbuminuria [OR = 9.93, 95 % CI (3.51–28.05), P < 0.001].

Conclusions

The findings suggest that plasma C4 may serve as a potential biomarker for the early stages of DN.

     

RNA Interference-Based Transgenic Maize Resistant to Maize Dwarf Mosaic Virus

Maize dwarf mosaic virus (MDMV) is a widespread pathogenic virus that causes serious loss of yield in maize (Zea mays). RNA interference (RNAi) triggered by hairpin RNA (hpRNA) transcribed from a transgenic inverted-repeat sequence is an effective way to defend against viruses in plants. In this study, an hpRNA expression vector containing a sense arm and an antisense arm of 150 bp separated by an intron of the maize actin gene was constructed to target the P1 protein (protease) gene of MDMV and used to transform Agrobacterium tumefaciens strain EHA105. The transformed Agrobacterium strain was used to transform maize embryonic calli isolated from immature embryos by an improved culture technique. In all, 46 plants were regenerated after stringent hygromycin B selection, and 18 of them were certified to be positive by PCR amplification. Of these positive plants, 13 were grown to produce offspring, and nine were identified by Southern blotting to have the transgene integrated with one or two copies. The resistance of three T₂ lines was evaluated in a field trial of dual MDMV inoculation in two environments and was found to be improved compared with the non-transformed control. The disease indexes of the transgenic plant lines h2, 13, and h1 were not significantly different from the highly resistant control line H9-21. The viral titers of the inoculated plants were detected by double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA), and the result was in accord with the resistance evaluated in the field trial. The addition of uniconazole S3307 (0.25 mg l⁻¹) and ABT root-promoting powder (0.5 mg l⁻¹) showed a significant improvement of hardening in regenerated plantlets, which were stronger and generated a better fibrous root system than the control. This improvement could facilitate the transgenic operation of maize.     

Lipid asymmetry in DLPC/DSPC-supported lipid bilayers: a combined AFM and fluorescence microscopy study

A fundamental attribute of cell membranes is transmembrane asymmetry, specifically the formation of ordered phase domains in one leaflet that are compositionally different from the opposing leaflet of the bilayer. Using model membrane systems, many previous studies have demonstrated the formation of ordered phase domains that display complete transmembrane symmetry; but there have been few reports on the more biologically relevant asymmetric membrane structures. Here we report on a combined atomic force microscopy and fluorescence microscopy study whereby we observe three different states of transmembrane symmetry in phase-separated supported lipid bilayers formed by vesicle fusion. We find that if the leaflets differ in gel-phase area fraction, then the smaller domains in one leaflet are in registry with the larger domains in the other leaflet and the system is dynamic. In a presumed lipid flip-flop process similar to Ostwald ripening, the smaller domains in one leaflet erode away whereas the large domains in the other leaflet grow until complete compositional asymmetry is reached and remains stable. We have quantified this evolution and determined that the lipid flip-flop event happens most frequently at the interface between symmetric and asymmetric DSPC domains. If both leaflets have identical area fraction of gel-phase, gel-phase domains are in registry and are static in comparison to the first state. The stability of these three DSPC domain distributions, the degree of registry observed, and the domain immobility have biological significance with regards to maintenance of lipid asymmetry in living cell membranes, communication between inner leaflet and outer leaflet, membrane adhesion, and raft mobility.     

Transcriptome analysis of age-related gain of callus-forming capacity in Arabidopsis hypocotyls

Callus-forming capacity is enhanced with hypocotyl maturity in Arabidopsis. However, the genetic regulation of age-related gain in capacity for callus formation is unclear. We used a gene expression microarray assay to characterize the underlying mechanisms during callus formation in young and mature hypocotyl explants of Arabidopsis. As expected, genes involved in photosynthesis and cell wall thickening showed altered expression during hypocotyl maturation. In addition, genes involved in cytokinin perception were enriched in mature hypocotyl tissues. Phytohormone-induced callus formation in hypocotyl explants was accompanied by increased expression of genes mainly related to the cell cycle, histones and epigenetics. The induction level of these genes was higher in mature hypocotyl explants than young explants during callus formation. We identified a number of genes, including those with unknown function, potentially involved in age-related gain in callus formation. Our results provide insight into the effect of hypocotyl age on callus formation. Altered cytokinin signaling components, cell cycle regulation and epigenetics may work in concert to lead to gain of callus-forming capacity in hypocotyls with age.     

Znf179 E3 ligase-mediated TDP-43 polyubiquitination is involved in TDP-43- ubiquitinated inclusions (UBI) (+)-related neurodegenerative pathology

Background

The brain predominantly expressed RING finger protein, Znf179, is known to be important for embryonic neuronal differentiation during brain development. Downregulation of Znf179 has been observed in motor neurons of adult mouse models for amyotrophic lateral sclerosis (ALS), yet the molecular function of Znf179 in neurodegeneration has never been previously described. Znf179 contains the classical C3HC4 RING finger domain, and numerous proteins containing C3HC4 RING finger domain act as E3 ubiquitin ligases. Hence, we are interested to identify whether Znf179 possesses E3 ligase activity and its role in ALS neuropathy.

Methods

We used in vivo and in vitro ubiquitination assay to examine the E3 ligase autoubiquitination activity of Znf179 and its effect on 26S proteasome activity. To search for the candidate substrates of Znf179, we immunoprecipitated Znf179 and subjected to mass spectrometry (MS) analysis to identify its interacting proteins. We found that ALS/ FTLD-U (frontotemporal lobar degeneration (FTLD) with ubiquitin inclusions)-related neurodegenerative TDP-43 protein is the E3 ligase substrate of Znf179. To further clarify the role of E3 ubiquitin ligase Znf179 in neurodegenerative TDP-43-UBI (ubiquitinated inclusions) (+) proteinopathy, the effect of Znf179-mediated TDP-43 polyubiquitination on TDP-43 protein stability, aggregate formation and nucleus/cytoplasm mislocalization were evaluated in vitro cell culture system and in vivo animal model.

Results

Here we report that Znf179 is a RING E3 ubiquitin ligase which possesses autoubiquitination feature and regulates 26S proteasome activity through modulating the protein expression levels of 19S/20S proteasome subunits. Our immunoprecipitation assay and MS analysis results revealed that the neuropathological TDP-43 protein is one of its E3 ligase substrate. Znf179 interactes with TDP-43 protein and mediates polyubiquitination of TDP-43 in vitro and in vivo. In neurodegenerative TDP-43 proteinopathy, we found that Znf179-mediated polyubiquitination of TDP-43 accelerates its protein turnover rate and attenuates insoluble pathologic TDP-43 aggregates, while knockout of Znf179 in mouse brain results in accumulation of insoluble TDP-43 and cytosolic TDP-43 inclusions in cortex, hippocampus and midbrain regions.

Conclusions

Here we unveil the important role for the novel E3 ligase Znf179 in TDP-43-mediated neuropathy, and provide a potential therapeutic strategy for combating ALS/ FTLD-U neurodegenerative pathologies.

     

Catechol-O-Methyltransferase Val158Met Polymorphism on Striatum Structural Covariance Networks in Alzheimer’s Disease

The catechol-O-methyltransferase enzyme metabolizes dopamine in the prefrontal axis, and its genetic polymorphism (rs4680; Val158Met) is a known determinant of dopamine signaling. In this study, we investigated the possible structural covariance networks that may be modulated by this functional polymorphism in patients with Alzheimer’s disease. Structural covariance networks were constructed by 3D T1 magnetic resonance imaging. The patients were divided into two groups: Met-carriers (n = 91) and Val-homozygotes (n = 101). Seed-based analysis was performed focusing on triple-network models and six striatal networks. Neurobehavioral scores served as the major outcome factors. The role of seed or peak cluster volumes, or a covariance strength showing Met-carriers > Val-homozygotes were tested for the effect on dopamine. Clinically, the Met-carriers had higher mental manipulation and hallucination scores than the Val-homozygotes. The volume-score correlations suggested the significance of the putaminal seed in the Met-carriers and caudate seed in the Val-homozygotes. Only the dorsal-rostral and dorsal-caudal putamen interconnected peak clusters showed covariance strength interactions (Met-carriers > Val-homozygotes), and the peak clusters also correlated with the neurobehavioral scores. Although the triple-network model is important for a diagnosis of Alzheimer’s disease, our results validated the role of the dorsal-putaminal-anchored network by the catechol-O-methyltransferase Val158Met polymorphism in predicting the severity of cognitive and behavior in subjects with Alzheimer’s disease.     

EphA2 receptor activation by monomeric Ephrin-A1 on supported membranes

The receptor tyrosine kinase EphA2 interacts with its glycosylphosphatidylinositol (GPI)-linked ephrin-A1 ligand in a juxtacrine configuration. The soluble ephrin-A1 protein, without its GPI membrane linker, fails to activate EphA2. However, preclustered ephrin-A1 protein is active in solution and has been frequently used to trigger the EphA2 receptor. Although this approach has yielded insights into EphA2 signaling, preclustered ligands bypass natural receptor clustering processes and thus mask any role of clustering as a signal regulatory mechanism. Here, we present EphA2-expressing cells with a fusion protein of monomeric ephrin-A1 (mEA1) and enhanced monomeric yellow fluorescent protein that is linked to a supported lipid bilayer via a nickel-decahistidine anchor. The mEA1 is homogeneously dispersed, laterally mobile, and monomeric as measured by fluorescence imaging, correlation spectroscopy, and photon counting histogram analysis, respectively. Ephrin-A1 presented in this manner activates EphA2 on the surface of MDA-MB-231 human breast cancer cells, as measured by EphA2 phosphorylation and degradation. Spatial mutation experiments in which nanopatterns on the underlying substrate restrict mEA1 movement in the supported lipid bilayer reveal spatio-mechanical regulation of this signaling pathway, consistent with recently reported observations using a synthetically cross-linked ephrin-A1 dimer.