An intersexual migratory (silver) longfinned New Zealand eel and its gonadal response to treatment with salmon pituitary homogenate

An intersexual adult New Zealand longfinned eel Anguilla dieffenbachii , displaying testicular tissue within its ovaries, is described. Gonad samples were collected at 0, 21, 42 (by biopsy) and 54 days (autopsy) after the start of an experiment that aimed to induce ovarian maturation by repeated treatment with 5 mg kg⁻¹ salmon pituitary homogenate (SPH). Very small patches of testicular tissue (almost always single seminiferous tubules), not visible macroscopically, were observed on all sampling dates. Treatment with SPH resulted in advanced stages of spermatogenesis in these seminiferous tubules, synchronous with the development of oocytes, which reached the migratory nucleus stage after 53 days. This finding is discussed in relation to current knowledge on sexual development in anguillid eels.     

Determination of plasma microRNA for early detection of gastric cancer

Gastric cancer is the fourth most prevalent malignancy worldwide and remains the second most common cause of cancer-related death globally. Understanding the molecular structure of gastric carcinogenesis might identify new diagnostic and therapeutic strategies for this disease. Thus, early detection of gastric cancer is a key measure to reduce the mortality and improve the prognosis of gastric cancer. There have recently been several reports that microRNAs (miRNAs) circulate in highly stable, cell-free forms in blood. Because serum and plasma miRNAs are relatively easy to access, circulating miRNAs also have great potential to serve as non-invasive biomarkers. Although a number of miRNAs associated with gastric cancer have been identified, the underlying mechanism of these miRNAs in tumorigenesis and tumor progression remains to be investigated. The purpose of this study is to identify the potential of serum miRNAs as biomarkers for early detection of gastric cancer patients. RNA was isolated using the High Pure miRNA Isolation Kit (Roche) following the manufacturer’s protocol. cDNA and preamplification protocols were obtained from the isolated plasma miRNAs. The BioMark? 96.96 Dynamic Array (Fluidigm Corporation) for real-time qPCR was used to simultaneously quantite the expression of 740 miRNAs. All statistical analyses were performed using the Biogazelle’s qbase PLUS 2.0 software. In this study, among 740 miRNAs that we analyzed only miR-195-5p was significantly (p < 0.05, fold changes = 13, 3) down-regulated in gastric cancer patients compared with control. We demonstrated that miR-195-5p is a novel tumor suppressor miRNA and may contribute to gastric carcinogenesis. The miRNA expression profile described in this study should contribute to future studies on the role of miRNAs in gastric cancer.     

Changes in steroid hormone profiles and ovarian histology during salmon pituitary-induced vitellogenesis and ovulation in female New Zealand longfinned eels, Anguilla dieffenbachii gray

To assess whether induced vitellogenesis in longfinned eels mimics that in naturally maturing conspecifics, female eels were artificially matured and steroid hormone status and oocyte cytology during oogenesis were evaluated. Successful induction of vitellogenesis was evident from the presence of yolk granules in the ooplasm of salmon pituitary homogenate (SPH)-injected, but not saline-, 17-hydroxyprogesterone-, and/or gonadotropin-releasing hormone-treated fish. In SPH-treated females, the migratory nucleus stage was reached after 33-53 days, followed by ovulation around 30 hours after induction of final maturation and ovulation. Only a portion of the germ cells matured, although resumption of vitellogenesis was seen in the majority of oocytes. In contrast, in ovaries of saline-injected controls, the most advanced oocytes were early vitellogenic. Atretic follicles were observed in ovaries of all eels, but abundance was greater in controls than in SPH-treated fish. SPH injections elevated plasma levels of estradiol-17beta and androgens, but not pregnenes, from within three days of treatment. Our results indicate that sex steroid levels in midvitellogenic hormone-treated females are similar to those in wild midvitellogenic females. In contrast, differences in yolk morphology of midvitellogenic follicles were seen between SPH-treated and wild females, especially in the second crop of midvitellogenic-sized oocytes measuring 300-400 microm in diameter. We discuss whether the observed differences affect egg quality, and perhaps explain the short life span of captive-bred eel larvae. J. Exp. Zool. 289:119-129, 2001.     

Efficient production of Arthromyces ramosus peroxidase by Aspergillus awamori

The heterologous production of Arthromyces ramosus peroxidase (ARP) was analysed in the filamentous fungus Aspergillus awamori under control of the inducible endoxylanase promoter. Secretion of active ARP was achieved up to 800 mg l(-1) in shake flask cultures. Western blot analysis showed that an rARP product of the correct molecular weight was produced. In contrast to several other studies about heterologous production of heme containing peroxidases, our results suggest that in A. awamori no heme limitation exists during overproduction of ARP.     

The production of antibody fragments and antibody fusion proteins by yeasts and filamentous fungi

In this review we will focus on the current status and views concerning the production of antibody fragments and antibody fusion proteins by yeasts and filamentous fungi. We will focus on single-chain antibody fragment production (scFv and V_HH) by these lower eukaryotes and the possible applications of these proteins. Also the coupling of fragments to relevant enzymes or other components will be discussed. As an example of the fusion protein strategy, the 'magic bullet' approach for industrial applications, will be highlighted.     

Otolith formation,microstructure and daily increment validation in juvenile perch Perca fluviatilis

The otoliths of laboratory‐reared larval and juvenile perch Perca fluviatilis of known age were analysed to determine the age of otolith formation and validate the formation of daily increments. There was a linear relationship between number of increments and age in days post‐hatching, although by 82 days post‐hatching daily increment counts underestimated actual age by an average of 5 days. Otolith dimensions in relation to standard length indicated allometric growth of otoliths until completion of yolk absorption, and isometric growth thereafter, up to 82 days post‐hatching.     

Fab fragments imprinted SPR biosensor for real-time human immunoglobulin G detection

F(ab) fragments imprinted surface plasmon resonance (SPR) chip was prepared for the real-time detection of human immunoglobulin G (IgG). In order to attach polymerization precursor on SPR chip, the SPR chip surface was modified with allyl mercaptan. F(ab) fragments of the IgG molecules were prepared by papain digestion procedure and collected by fast protein liquid chromatography (FPLC) system using Hi-Trap_r Protein A FF column. The collected F(ab) fragments were complexed with histidine containing specific monomer, N-methacryloyl-l-histidine methyl ester (MAH). Molecular imprinted polymeric nanofilm was prepared on SPR chip in the presence of ethylene glycol dimethacrylate and 2-hydroxyethylmethacrylate. The template molecules, F(ab) fragments, were removed from the polymeric nanofilm using 1M NaCl solution (pH: 7.4, phosphate buffer system). The molecular imprinted SPR chip was characterized by contact angle, atomic force microscopy and Fourier transform infrared spectroscopy. By the real-time IgG detection studies carried out using aqueous IgG solutions in different concentrations, the kinetics and isotherm parameters of the molecular imprinted SPR chip-IgG system were calculated. To show selectivity and specificity of the molecular imprinted SPR chip, competitive kinetic analyses were performed using bovine serum albumin (BSA), IgG, F(ab) and F(c) fragments in singular and competitive manner. As last step, IgG detection studies from human plasma were performed and the measured IgG concentrations were well matched with the results determined by enzyme-linked immunosorbent assay (ELISA). The results obtained with the molecular imprinted SPR chip were well fitted to Langmuir isotherm and the detection limit was found as 56 ng/mL. In the light of the results, we can conclude that the proposed molecular imprinted SPR chip can detect IgG molecules from both aqueous solutions and complex natural samples.     

Ubiquitously expressed hematological and neurological expressed 1 downregulates Akt-mediated GSK3β signaling, and its knockdown results in deregulated G2/M transition in prostate cells

As the molecular mechanism of β-catenin deregulation is not well understood, and stabilized β-catenin is known to translocate into the nucleus and activate genes for proliferation, a novel regulatory factor, hematological and neurological expressed 1 (HN1), for Akt-GSK3β-β-catenin axis is reported here. In our studies, HN1 gene structure was characterized. HN1 expression was found to be epidermal growth factor-responsive in PC-3 cells, and protein expression was also upregulated in PC-3 and LNCaP but not in DU145 cells. Additionally, HN1 was found to be downregulated by the specific AKT inhibitor wortmannin but not with PI3K or MAPK inhibitors, LY294002 and PD98059, respectively, in PC-3 and MCF-7 cells. Further, siRNA-mediated knockdown of HN1 resulted in considerable increase in Akt((S473)) and GSK3β((S9),(Y216)) phosphorylations; moreover, subsequent accumulation of β-catenin, increase in c-myc expression, and nuclear accumulation of cyclin D1 were observed in PC-3 cells. Knockdown of HN1 also resulted in prolongation of G(1) phase in cell cycle, increasing tetraploidy, presumably because of cells escaping from abnormal mitosis in PC-3 cells. Consistently, overexpression of HN1 reversed the cell-cycle-specific observations, resulted in accumulation of cells in G(2)/M, and reduced the proliferation rate, which were investigated using flow cytometry and methylthiazol tetrazolium assays. As activating mutations of β-catenin have been demonstrated in late-stage tumors, and β-catenin stabilization was correlated with poor prognosis in previous reports, epidermal growth factor-upregulated HN1 expression might have a role in deregulating the AKT-GSK3β((S9))-mediated signaling as a novel compensating mechanism.