Aminoglycosides are efficient reagents to induce readthrough of premature termination codon in mutant B4GALNT1 genes found in families of hereditary spastic paraplegia

The readthrough of premature termination codon (PTC) by ribosome sometimes produces full-length proteins. We previously reported a readthrough of PTC of glycosyltransferase gene B4GALNT1 with hereditary spastic paraplegia (HSP). Here we featured the readthrough of B4GALNT1 of two mutants, M4 and M2 with PTC by immunoblotting and flow cytometry after transfection of B4GALNT1 cDNAs into cells. Immunoblotting showed a faint band of full-length mutant protein of M4 but not M2 at a similar position with that of wild-type B4GALNT1. AGC sequences at immediately before and after the PTC in M4 were critical for the readthrough. Treatment of cells transfected with mutant M4 cDNA with aminoglycosides resulted in increased readthrough of PTC. Furthermore, treatment of transfectants of mutant M2 cDNA with G418 also resulted in the induction of readthrough of PTC. Both M4 and M2 cDNA transfectants showed increased/induced bands in immunoblotting and GM2 expression in a dose-dependent manner of aminoglycosides. Results of mass spectrometry supported this effect. Here, we showed for the first time the induction and/or enhancement of the readthrough of PTCs of B4GALNT1 by aminoglycoside treatment, suggesting that aminoglycosides are efficient for patients with HSP caused by PTC of B4GALNT1, in which gradual neurological disorders emerged with aging.

Deteksi Koi Herpesvirus (KHV) pada Ikan Nila (Oreochromis niloticus) yang Diinfeksi secara Buatan [ Detection of Koi Herpesvirus (KHV) in Nile Tilapia (Oreochromis niloticus) Infected by Artificially Infection ]

Koi Herpesvirus (KHV) was formerly known as Cyprinid herpesvirus-3 (CyHV-3) and carp nephritis interstitial and necrosis gill virus (CNGV) is a virus that infects common carp and koi (Cyprinus carpio and C. carpio koi) in farmed and wild population. KHV cause disease at a temperature of 18-25 °C with mortality rate of 80-90 % in fry and adult fish. Currently KHV also detected in tilapia from the results of monitoring in the field. The presence of KHV in tilapia can occur as a result of maintenance in cages adjacent to the infected carp. KHV diagnostic method currently based on case definition and PCR (Polymerase Chain Reaction) detection. The basic concept of PCR is one DNA molecule is used to produce two copies, then four, then eight and so forth through multiplication by polymerase. PCR results sometimes indicated the presence of a faint band caused a low amount of virus, so it is necessary to investigate the presence of KHV DNA in tilapia using different doses of infection. This study aimed to determine the KHV infectivity in nile tilapia were infected by artificially infection and determine dose KHV infection that can infect nile tilapia. The study design used true experimental with with the presentation of descriptive data. Dose of viral infection are 1 ID50, 10 ID50, 100 ID50 and 1000 ID50. The results showed that no clinical symptoms of KHV infected in nile tilapia. The results of electrophoresis of PCR products showed that the mucus of nile tilapia were infected with a 1000 ID50 immersion dose showed thin bands. The same results are also shown in the gill of nile tilapia infected by gill spray at 1000 ID50 dose. Fish were infected by injection, KHV was not detected in mucus, gill, kidney and liver. The results above show nile tilapia cannot be infected by KHV on various treatment

Photoluminescence and Thermoluminescence of Phosphate Glasses Doped with Dy3+ and Containing Silver Nanoparticles

Phosphate glasses doped with Dy[Formula: see text] ions and containing silver nanoparticles (SNPs) were synthesized in the present work. We report photoluminescence characterization by absorption and emission spectra. The effect of Ag concentration on the thermoluminescence (TL) glow curves was studied. The scanning electron microscopy (SEM) shows the formation of SNP. Absorption spectra of the samples show the influence of the SNP in the bands 350[Formula: see text]nm at 425[Formula: see text]nm associated with the Dy[Formula: see text], in the same spectra we can see the bands 750, 800, 875, 1098, 1278[Formula: see text]nm and 1675[Formula: see text]nm belonging to the Dy[Formula: see text]. Emission spectra show two prominent bands at 480[Formula: see text]nm and 574[Formula: see text]nm and one faint band at 665[Formula: see text]nm corresponding to 4F[Formula: see text]H[Formula: see text], 4F[Formula: see text]H[Formula: see text] and 4F[Formula: see text]H[Formula: see text] transitions, respectively. All bands under 364[Formula: see text]nm pumping, and the fluorescence in the 550[Formula: see text]nm and 590[Formula: see text]nm spectral range enhanced four times. The Commission Internationale de 1’Eclairage (CIE) color coordinates were evaluated from the emission spectra to simulate white light emission from the phosphate glasses. The photostability of the samples was also studied in the UVA (315–403[Formula: see text]nm) and UVB (280–315[Formula: see text]nm) ranges. TL due to ultraviolet radiation (UVR) was studied; the glow curves show significant dependence of the TL intensity with the increment of SNPs in the samples. These results show the phosphate glasses doped with Dy[Formula: see text] and containing SNP as a potential candidate have to be used in solid-state illumination and retrospective dosimetry.

Characterization of human phosphoserine aminotransferase involved in the phosphorylated pathway of l-serine biosynthesis

In the present study, we first report two forms of human phosphoserine aminotransferase (PSAT) cDNA (HsPSATα and HsPSATβ). HsPSATα has a predicted open reading frame comprising 324 amino acids, encoding a 35.2 kDa protein (PSATα), whereas HsPSATβ consists of an open reading frame comprising 370 amino acids that encodes a 40 kDa protein (PSATβ). PSATα is identical with PSATβ, except that it lacks 46 amino acids between Val290 and Ser337 of PSATβ, which is encoded by the entire exon 8 (138 bp). Both PSATα and PSATβ can functionally rescue the deletion mutation of the Saccharomyces cerevisiae counterpart. Reverse transcriptase–PCR analysis revealed that the expression of PSATβ mRNA was more dominant when compared with PSATα mRNA in all human cell lines tested. PSATβ was easily detected in proportion to the level of mRNA; however, PSATα was detected only in K562 and HepG2 cells as a very faint band. The relative enzyme activity of glutathione S-transferase (GST)–PSATβ expressed in Escherichia coli appeared to be 6.8 times higher than that of GST–PSATα. PSAT mRNA was expressed at high levels (approx. 2.2 kb) in the brain, liver, kidney and pancreas, and very weakly expressed in the thymus, prostate, testis and colon. In U937 cells, the levels of PSAT mRNA and protein appeared to be up-regulated to support proliferation. Accumulation of PSAT mRNA reached a maximum in the S-phase of Jurkat T-cells. These results demonstrate that although two isoforms of human PSAT can be produced by alternative splicing, PSATβ rather than PSATα is the physiologically functional enzyme required for the phosphorylated pathway, and indicate that the human PSAT gene is regulated depending on tissue specificity as well as cellular proliferation status with a maximum level expression in the S-phase.

Testosterone and dihydrotestosterone regulate AUF1 isoforms in a tissue-specific fashion in the mouse

.—The sex difference in the metabolism of certain mRNAs in the murine submaxillary gland (SMG) prompted us to determine whether androgens regulate the expression of any of the four isoforms of AUF1, proteins that bind differentially to AU-rich RNA. We found that cytosol from female SMGs contains two major isoforms (p45 and p40), whereas cytosol from male SMGs contains a prominent p37 and a weaker p42. Injecting female mice with testosterone decreases p45 levels by 81% after 7 days ( P < 0.05, n = 4), whereas p42 and p37 increase 74 and 449% at 7 days ( P < 0.05, n = 4, for both). Orchiectomy, conversely, decreases p37 levels in the male SMG by 91% ( P < 0.006) while increasing p45 5-fold and p40 2.5-fold ( P < 0.05, n = 5 for both). Both male and female kidney cytosol contains a prominent p37 and a faint band of ∼42 kDa, but neither shows a significant change when circulating androgen levels are altered. Dihydrotestosterone (DHT) changes the pattern of AUF1 isoforms in female SMG cytosol more rapidly than does testosterone. Nuclear extracts from female SMG contain predominantly p45, and DHT decreases its level slightly (35%, P < 0.05 at 24 h). Polysomal extracts from female SMG contain p45 and p42, and DHT increases p45 levels 58% ( P < 0.02, n = 6) at 24 h. In certain nonreproductive tissues, androgens may differentially regulate AUF1 isoform levels to modulate the metabolism of AU-rich mRNAs posttranscriptionally.

Detection of serum insulin-like growth factor binding proteins on Western ligand blots by biotinylated IGF and enhanced chemiluminescence

A novel procedure for the detection of IGF binding capacity of IGFBPs on Western ligand blots (WLB) was developed using biotinylated IGFs as probes. The biotinylated IGF-IGFBP complexes were visualized by streptavidin-horseradish peroxidase and enhanced chemiluminescence (ECL). The procedure was found to be faster and more efficient than the conventional method with iodinated IGFs. In normal human serum a predominant doublet at 38-42 kDa and five smaller bands at 35, 34, 30, 28 and 24 kDa were detected by both methods, whereas two additional bands at 26 and 16 kDa became visible with the ECL method. In pregnancy serum only one single faint band at 30 kDa could be detected by the iodinated method. In contrast, the ECL method revealed five other bands at 42, 34, 28, 26 and 16 kDa. Besides the 38-42 kDa doublet, the 30 and 16 kDa bands reacted strongly with anti-IGFBP-3 antibodies in Western immunoblotting (WIB) and therefore were related to IGFBP-3 fragments. The technical advantages of this ECL method include an extremely short exposure time to the radiographic film and a long stability of the probe. In addition, the ECL method is a non-radioactive method, making radioprotection and radioactive waste removal unnecessary.

Preparation and characterization of an antibody specific to the rat dopamine D2 receptor

ABSTRACT Antibodies specific to the dopamine D2 receptor have been raised in rabbits using synthetic peptides. The resulting antiserum was sensitive to picogram quantities of peptide as measured by enzyme-linked immunoassay and was shown to have a 33% cross-reactivity with partially purified D2 receptor protein. No detectable cross-reactivity with similarly prepared fungal membranes was observed. D2 receptor preparations from normal rat pituitary cells were used in Western blot analysis. Bands of Mr = 95 000 and 34 000 were detected in these preparations with a third faint band at 120 000. These correspond to the pituitary D2 receptor. Journal of Endocrinology (1992) 134, 227–233

Isolation and partial characterization of host cell surface agglutinin and its role in attachment of a biotrophic mycoparasite

Cell surface proteins obtained by alkaline extraction from isolated cell walls of Mortierella pusilla and M. candelabrum, host and nonhost, respectively, of the mycoparasite Piptocephalis virginiana, were tested for their ability to agglutinate mycoparasite spores. The host cell wall protein extract had a high agglutinating activity (788 agglutination units/mg) compared with that of the nonhost extract (21 agglutination units/mg). Sodium dodecyl sulfate – polyacrylamide gel electrophoresis of the crude extract of the host revealed four bands, a, b, c, and d, with respective Mr of 117 000, 100 000, 85 000 and 64 000; these bands except for a faint band c, were absent from the nonhost surface. Deletion of proteins b or c from the crude protein extract of the host significantly reduced its agglutinating activity. Proteins b and c, purified by a series of procedures, were shown to be glycoproteins with glucose and N-acetylglucosamine as major saccharides. The agglutinating activity of a mixture of pure proteins b and c was over 500 times that of either glycoprotein alone, suggesting an involvement of both glycoproteins in the agglutination process. Further characterization showed that the two glycoproteins were heat-resistant with respect to their agglutinin function, which could be totally inhibited by three sugars: arabinose, glucose and N-acetyglucosamine. It is suggested that glycoproteins b and c are the two subunits of a carbohydrate-binding agglutinin present at the host cell surface and involved in agglutination and attachment of the mycoparasite germ tubes. Key words: agglutinin, attachment, cell surface, sugars, glycoproteins, mycoparasitism.

Regulation of mouse mammary-gland γ-glutamyltranspeptidase mRNA during pregnancy, lactation and weaning

The level of gamma-glutamyltranspeptidase (GGT) activity and of its mRNA were determined in the mouse mammary gland during pregnancy, lactation and weaning. The GGT activity, which is very low in the virgin-mouse mammary gland (5 munits/mg of protein), increases progressively during pregnancy (3-fold), reaches its maximum at the onset of lactation (8-fold) and returns rapidly to basal level at weaning. Although no GGT-specific mRNA is detected in the virgin-mouse mammary gland, a single faint band of 2.2 kb in size is found during pregnancy. During lactation, an additional mRNA of 2.4 kb in size appears, and the level of both mRNAs is higher. This high level of mRNA persists during weaning as well. Southern-blot analysis of mouse mammary-gland DNA provides convincing evidence that there is only one gene which codes for the two mRNAs. The present study provides the first evidence for a physiological regulation of the two GGT mRNAs in the same tissue.