Unique structure in the intergenic and 5' external transcribed spacer of the ribosomal RNA gene from the pea aphid Acyrthosiphon pisum.

We analyzed the DNA sequence of the 5' external transcribed spacer (ETS) and part of the intergenic transcribed spacer (IGS) of the aphid ribosomal RNA gene (rDNA). The 5' ETS of aphid rDNA consists of 843 nucleotides with a G/C content of 69 mol/100 mol, far higher than that of any other known 5' ETS for insect rDNA. The IGS of aphid rDNA contained a characteristic array of repeated sequences of 247 nucleotides. The repeated sequences were identical. It was shown that the number of the repeating sequence is heterogeneous.     

Targeted mass spectrometric approach for biomarker discovery and validation with nonglycosylated tryptic peptides from N-linked glycoproteins in human plasma

A simple mass spectrometric approach for the discovery and validation of biomarkers in human plasma was developed by targeting nonglycosylated tryptic peptides adjacent to glycosylation sites in an N-linked glycoprotein, one of the most important biomarkers for early detection, prognoses, and disease therapies. The discovery and validation of novel biomarkers requires complex sample pretreatment steps, such as depletion of highly abundant proteins, enrichment of desired proteins, or the development of new antibodies. The current study exploited the steric hindrance of glycan units in N-linked glycoproteins, which significantly affects the efficiency of proteolytic digestion if an enzymatically active amino acid is adjacent to the N-linked glycosylation site. Proteolytic digestion then results in quantitatively different peptide products in accordance with the degree of glycosylation. The effect of glycan steric hindrance on tryptic digestion was first demonstrated using alpha-1-acid glycoprotein (AGP) as a model compound versus deglycosylated alpha-1-acid glycoprotein. Second, nonglycosylated tryptic peptide biomarkers, which generally show much higher sensitivity in mass spectrometric analyses than their glycosylated counterparts, were quantified in human hepatocellular carcinoma plasma using a label-free method with no need for N-linked glycoprotein enrichment. Finally, the method was validated using a multiple reaction monitoring analysis, demonstrating that the newly discovered nonglycosylated tryptic peptide targets were present at different levels in normal and hepatocellular carcinoma plasmas. The area under the receiver operating characteristic curve generated through analyses of nonglycosylated tryptic peptide from vitronectin precursor protein was 0.978, the highest observed in a group of patients with hepatocellular carcinoma. This work provides a targeted means of discovering and validating nonglycosylated tryptic peptides as biomarkers in human plasma, without the need for complex enrichment processes or expensive antibody preparations.     

Regulatory role of osteopontin in malignant transformation of endometrial cancer

Osteopontin (OPN) involves in the tumor-promoting or metastasis in human endometrial cancer. Depletion of OPN gene expression in endometrial cancer cells was significantly decreased in cell viability and the cells undergo apoptotic cell death. The status of OPN in THESC, RL95, Hec1A and Ishikawa cell lines were analyzed by RT-PCR and western blot. After OPN-siRNA transfection, mRNA and protein expression levels of OPN were determined in Hec1A and Ishikawa cells. Cell proliferation and cell cycle distribution were observed by MTT and flow cytometry analysis. DNA fragmentation assay was used to measure cell apoptosis. Cell migration was assessed by wound healing assay. Depletion of OPN gene expression in endometrial cancer cell lines (Hec1A and Ishikawa cells) reproducibly changed their ability of proliferation. Concomitant changes were seen in the expression of OPN binding cell surface receptors, cell cycle-regulatory genes, cell invasion and colony formation nature of the tumor cells. Decreased colonizing potential in the absence of OPN was reversed in the presence of recombinant OPN. Inhibition of anchorage-independent growth was observed in the presence of metabolic inhibitors of the PI3K, Src and integrin signaling cascades, which was ameliorated in the presence of exogenously added OPN. Our result showed the role of OPN in endometrial cancer, in particular on the malignancy-promoting aspects of OPN that may pave way for new approaches to the clinical management of endometrial cancer.     

A Simple Clearing Method for Improved Visualization of lacZ Expression in Fish Larvae

The protocol described in this report provides a simple and reliable method for improved visualization of lacZ expression upon X-gal staining. The simple treatments of KOH-H2O2 and glycerol removed all pigment from the whole body and made the demelanized fry transparent without any structural damage. Based on this method, the exogenous expression of lacZ could be easily identified and distinguished from endogenous background activity in pigmented transgenic fry.     

Harvesting of microalgae using flocculation combined with dissolved air flotation

This study explored efficient methods of harvesting the Tetraselmis sp. KCTC12236BP using flocculation and dissolved air flotation. Concentration ranges of flocculation agents were optimized using jar tests (batch flocculation experiments) using inorganic (aluminum sulfate, ferric sulfate) and organic (chitosan) flocculants in a pH range of 4 ～ 10. The optimal dosage and pH level were 1.2 g/L and pH 5 ～ 6 for aluminum sulfate, 0.7 g/L and pH 4 ～ 8 for ferric sulfate, and 5.0 mg/mL and pH 7 ～ 8 for chitosan. The highest harvesting efficiency achieved with each of the four compounds was 85.6, 92.6, 93, and 91.3%, respectively.     

Peripubertal requirement of Tsg101 in maintaining the integrity of membranous structures in mouse oocytes

ObjectiveAs a component of Endosomal Sorting Complex Required for Transport (ESCRT) complex I, the tumor susceptibility gene 101 (Tsg101) carries out multiple functions. In this work, we report that oocyte‐specific deletion of tumor susceptibility gene 101 (Tsg101) leads to age‐dependent oocyte demise in mice.Materials and Method Tsg101 floxed mice (Tsg101 ^f/f ) were bred with Zp3 ^cre transgenic mice to examine oocyte‐specific roles of Tsg101. Multiple cellular and molecular biological approaches were taken to examine what leads to oocyte demise in the absence of Tsg101.ResultsThe death of oocytes from Zp3 ^cre /Tsg101 ^f/f (Tsg101 ^d/d thereafter) mice showed a strong correlation with sexual maturation, as gonadotropin‐releasing hormone antagonist injections improved the survival rate of oocytes from 5‐week‐old Tsg101 ^d/d mice. Maturation of oocytes from prepubertal Tsg101 ^d/d mice proceeded normally, but was largely abnormal in oocytes from peripubertal Tsg101 ^d/d mice, showing shrinkage or rupture. Endolysosomal structures in oocytes from peripubertal Tsg101 ^d/d mice showed abnormalities, with aberrant patterns of early and late endosomal markers and a high accumulation of lysosomes. Dying oocytes showed plasma membrane blebs and leakage. Blockage of endocytosis in oocytes at 4°C prevented cytoplasmic shrinkage of oocytes from Tsg101 ^d/d mice until 9 h. The depletion of tsg‐101 in Caenorhabditis elegans increased the permeability of oocytes and embryos, suggesting a conserved role of Tsg101 in maintaining membrane integrity.ConclusionsCollectively, Tsg101 plays a dual role in maintaining the integrity of membranous structures, which is influenced by age in mouse oocytes.

Tsg101 deficiency in mouse oocytes leads to a complex phenotype involving the plasma membrane (PM). These oocytes are normal until mice reach five weeks of age when oocytes begin to show PM rupture, PM blebbing, and cytoplasmic shrinkage. PM blebbing and cytoplasmic shrinkage can be postponed with a general endocytosis block, but death ensues. In both mouse and in Caenorhabditis elegans, Tsg101 plays a crucial role in maintaining the integrity of PM.