Springer Science and Business Media LLC

Genetic improvement of soybean oil content depends on in-depth study of the glycerolipid biosynthesis pathway. The first acylation reaction catalysed by glycerol-3-phosphate acyltransferase (GPAT) is the rate-limiting step of triacylglycerol biosynthesis. However, the genes encoding GPATs in soybean remain unknown. We used a novel yeast genetic complementation system and seed-specific heterologous expression to identify GmGPAT activity and molecular function in glycerolipid biosynthesis. Sixteen GmGPAT genes were cloned by reverse transcription-PCR for screening in yeast genetic complementation. The results showed that GmGPAT9-2 could restore the conditional lethal double knockout mutant strain ZAFU1, and GmGPAT1-1 exhibited low acyltransferase activity in serial dilution assays. In addition, the spatiotemporal expression pattern of GmGPAT9-2 exhibited tissue specificity in leaves, flowers and seeds at different developmental stages. Furthermore, both the proportion of arachidic acid and erucic acid were significantly elevated in Arabidopsis thaliana transgenic lines containing the seed-specific GmGPAT9-2 compared wild type, but the oil content was not affected. Together, our results provide reference data for future engineering of triacylglycerol biosynthesis and fatty acid composition improvement through GPATs in soybean.

A primary obstacle in age-related hearing loss (ARHL) study is the lack of accelerated senescent models in vitro that explore the precise underlying mechanism in different types of ARHL. The damage to strial marginal cells (SMCs) is a subset of strial presbycusis-associated pathological changes. We aimed to establish a D-galactose (D-gal)-induced SMCs senescent model and study the effect of deacetylase sirtuin 1 (SIRT1) on presbycusis in vitro. SMCs from C57BL/6J neonatal mice were cultured and treated with D-gal to establish accelerated senescent models. And then D-gal-induced SMCs were transfected with adenovirus (Ad)-SIRT1-GFP or Ad-GFP. Oxidative stress and mitochondrial DNA (mtDNA) damage were determined by histological analysis or RT-PCR. Western blotting (WB) and RT-PCR were used to evaluate protein and mRNA levels of superoxide dismutase 2 (SOD2) and SIRT1, respectively. Additionally, apoptosis was investigated by WB and TUNEL staining. D-gal-induced SMCs exhibited several characteristics of senescence, including increased the level of 8-hydroxy-2′-deoxyguanosine, which is a marker of DNA oxidative damage, and elevated the amount of mtDNA 3860-bp deletion, which is a common type of mtDNA damage in the auditory system of mice. SIRT1 overexpression effectively inhibited these changes by upregulating the level of SOD2, thereby inhibiting cytochrome c translocation from mitochondria to cytoplasm, inhibiting cell apoptosis, and ultimately delaying aging in the D-gal-induced senescent SMCs. Altogether, the evidence suggests that the D-gal-induced SMCs accelerated aging model is successfully established, and SIRT1 overexpression protects SMCs against oxidative stress by enhancing SOD2 expression in ARHL.

The insulin-like growth factor (IGF) signaling pathway has an important role in many cancers, including esophageal cancer (EC). IGF-binding protein 7 (IGFBP7) is one of the proteins in this signaling pathway, and its role in cancer has not yet been fully clarified. In the present study, we evaluated the clinical relevance of IGFBP7 methylation status and mRNA expression in EC patients compared to healthy controls. We also investigated whether IGFBP7 methylation status affects mRNA expression. The study comprised 100 EC patients and 105 healthy controls. Methylation specific PCR (MSP) was used to examine IGFBP7's promoter methylation and real-time quantitative reverse transcription PCR (qRT-PCR) was used to assess IGFBP7 mRNA expression. The IGFBP7 promoter methylation was significantly higher in controls than in EC patients (p < 0.05). IGFBP7 mRNA expression was significantly lower in EC patients compared to controls, especially in those over 55 years old (p < 0.0001). The globulin level and reflux were significantly higher in IGFBP7-unmethylated patients compared to IGFBP7 methylated patients (p = 0.01). In EC patients, however, there was no significant relationship between IGFBP7 mRNA expression and methylation in the peripheral blood (p = 0.33). In addition, neither IGFBP7 mRNA expression nor methylation were shown to be linked with survival (p > 0.05). Our study indicated that promoter unmethylation and mRNA expression of the IGFBP7 promoter in peripheral blood could be different biomarkers for EC. Furthermore, unmethylation of the IGFBP7 promoter in EC patients was associated with reflux and elevated globulin levels. More studies with a larger number of cases is needed to confirm this association.

We show that the Th/ To ribonucleoprotein is defined by (i) the co-immunoprecipitation of two RNAs, (ii) the co-immunoprecipitation of four major polypeptides and (iii) the quantitative immune recognition of both RNase P and RNase MRP. No serum was found that recognizes either one of these two enzymes exelusively. The specific co-immunoprecipitation of RNase MRP and RNase P by all Th/ To ribonucleoprotein autoantibodies indicates that the anti-Th/ To autoimmune response is directed against both enzymes in a quantitatively indistinguishable manner. Thus the Th/ To ribonucleoprotein is defined by RNase P and RNase MRP.

The present study reports the complete sequences of Aquaporin 1 (AQP1) gene and partial sequences of genes, Sodium/potassium-transporting ATPase subunit alpha-1 (Na/K-ATPase α1 subunit), Osmotic Stress Transcription Factor 1 (OSTF1), Transcription Factor II B (TFIIB), Heat Shock Cognate 71 (HSC71) and Heat Shock Protein 90 (HSP90) obtained from mRNA and genomic DNA of Etroplus suratensis. They are candidate genes involved in stress responses of fishes. AQP1 gene was 2163 bp long. Its mRNA sequence has 55 bp 5′ UTR, 783 bp open reading frame (ORF), 119 bp 3′ UTR, three intronic regions and 90% identity with AQP1 of Oreochromis niloticus. The partial Na/K-ATPase α1subunit gene obtained 5998 bp length with an ORF of 2213 bp and 12 intronic regions. The partial OSTF1, TF IIB, HSC71 and HSP90 mRNA sequences obtained were 1473 bp, 587 bp, 1708 bp and 151 bp in length respectively. All the genes showed a high sequence similarity with respective genes reported from fishes. Comparison of AQP1 and Na/K-ATPase α1 genomic DNA sequence of E. suratensis collected from different water system showed two type of AQP1 with one synonymous mutation in exon-1 and higher sequence difference in intronic regions (including addition, deletion, transition and transversion mutations) with few synonymous and non-synonymous mutations in the exons of Na/K-ATPase α1. The sequence information of these major candidate genes involved in stress responses will help in further studies on population genetics, adaptive variations and genetic improvement programs of this cichlid species having aquaculture, ornamental and evolutionary importance.

A novel bacterial strain with high cellulase activity (2.82 U/ml) was isolated, and then identified by its morphological character and 16S rRNA sequence, and named Bacillus subtilis strain I15. The extracellular thermostable cellulase exhibited the maximum activity at 60°C and pH 6.0. It was very stable since more than 90% of original CMCase activity was maintained at 65°C after incubation for 2 h. The cellulase gene, celI15, was cloned and extracellularly expressed by Escherichia coli BL21 (DE3), which encoded the extracellular protein of about 52 kDa. The extracellular activity of CelI15 from E. coli BL21 was up to about 6.78 U/ml, and all the other properties were almost the same as that from the wild-type strain.

The ATP-binding cassette (ABC) superfamily is a larger protein family with diverse physiological functions in all kingdoms of life. We identified 53 ABC transporters in the silkworm genome, and classified them into eight subfamilies (A–H). Comparative genome analysis revealed that the silkworm has an expanded ABCC subfamily with more members than Drosophila melanogaster, Caenorhabditis elegans, or Homo sapiens. Phylogenetic analysis showed that the ABCE and ABCF genes were highly conserved in the silkworm, indicating possible involvement in fundamental biological processes. Five multidrug resistance-related genes in the ABCB subfamily and two multidrug resistance-associated-related genes in the ABCC subfamily indicated involvement in biochemical defense. Genetic variation analysis revealed four ABC genes that might be evolving under positive selection. Moreover, the silkworm ABCC4 gene might be important for silkworm domestication. Microarray analysis showed that the silkworm ABC genes had distinct expression patterns in different tissues on day 3 of the fifth instar. These results might provide new insights for further functional studies on the ABC genes in the silkworm genome.

The regulation of mitochondrial respiration in the intact heart may differ from that of isolated mitochondria if intracellular diffusion is restricted. Here we consider which factors may hinder diffusion in vivo and, based on computational analysis, design a reverse engineering approach to estimate the role of diffusional resistance in mitochondrial regulation from an experiment on the intact heart. Computational analysis of respiration measurements on skinned heart fibers shows that the outer mitochondrial membrane does not hinder diffusion enough to cause ADP gradients of tens of micromolars. A diffusion model further shows that the mesoscale structure of the myofibrillar space also does not hinder diffusion appreciably. However, ADP gradients are suggested by the measured activation time of oxidative phosphorylation and may be caused by diffusion restriction of other intracellular structures or the in vivo microstructure of networks of physically interacting proteins. Based on computational modeling we propose an experiment on the intact heart that allows to estimate the effective diffusion restriction between ATP producing and consuming sites in the cardiac cell.

Melia dubia Cav. is a fast-growing multipurpose tree suitable for agroforestry and has been widely cultivated for wood-based industries, particularly pulp and paper production. Despite its high economic value in India, there is a lack of information regarding the molecular mechanism driving its fast-growth. Therefore, this study aimed to elucidate the molecular mechanisms responsible for fast-growth by expression analysis of selective candidate genes. Initially, growth traits were assessed, including tree height and diameter at breast height (DBH), across three different ages (one-year-old, two-year-old, and three-year-old) of M. dubia plantations. Tree volume based on tree height and DBH, was also calculated. The analysis of annual tree height increment revealed that the second-year plantation exhibited the higher increment, followed by first and third years. In contrast, DBH was maximum in third-year plantation, followed by the second and first years. Similarly, annual tree volume increment showed a similar trend with DBH that maximum in the third year, followed by second and first years. Furthermore, a differential gene expression analysis was performed using qRT-PCR on four genes such as Phloem Intercalated with Xylem (PXY), Clavata3/Embryo Surrounding Region-Related 41 (CLE41), 1-aminocyclopropane-1-carboxylic acid synthase (ACS-1) and Hemoglobin1 (Hb1) for downstream analysis. The relative gene expression showed up-regulation of CLE41, ACS-1, and Hb1 genes, while the PXY gene was downregulated across the tree ages. Interestingly, a positive association was observed between tree growth and the expression of the selected candidate genes. Our results pave the way for further research on the regulatory mechanisms of genes involved in fast-growth and provide a basis for genetic improvement of Melia dubia.

The skin is constantly exposed to various external stimuli including humidity variations. Low humidity affects skin properties such as decreased water content of the stratum corneum, reduced skin elasticity, and itching. However, the effects of humidity on the skin cells are not completely understood. This study aimed to investigate how low humidity affects keratinocytes of the skin. In the present study, the effects of dry environment on the gene expression profile of epidermal keratinocytes were demonstrated using a three-dimensional skin model (3D-skin), composed of keratinocytes. Exposure of 3D-skin to low humidity (relative humidity ~ 10%) increased the expression levels of various genes, including those related to signal transduction and immune system. Accordingly, p38 mitogen-activated protein kinase (MAPK) signaling in keratinocytes of the 3D-skin was activated in response to low humidity for 30 min. Additionally, several chemokines, such as chemokine (C-X-C motif) ligand 1 (CXCL1) and Chemokine (C-C motif) ligand 20 (CCL20), were up regulated after 3 h of exposure to low humidity. We hypothesize that increased chemokine production may affect the immune system of the whole skin through chemoattractants. Our findings imply that keratinocytes sense low humidity and resultant activation of some cell-signaling pathways leads to variations in gene expression profiles including various chemokines. We provide evidence that keratinocytes adapt to external humidity variations.