Isolation of new CHO cell mutants defective in CMP-sialic acid biosynthesis and transport

Sialic acid is a sugar typically found at the N-glycan termini of glycoproteins in mammalian cells. Lec3 CHO cell mutants are deficient in epimerase activity, due to a defect in the gene that encodes a bifunctional UDP-GlcNAc 2-epimerase/ManNAc kinase (GNE). Sialic acid modification on the cell surface is partially affected in these cells. We have mutagenized Lec3 CHO cells and isolated six mutants (termed C2m) deficient in the cell surface expression of polysialic acid (PSA). Mutant C2m9 was partially defective in expression of cell-surface PSA and wheat germ agglutinin (WGA) binding, while in the other five mutants, both cell-surface PSA and WGA binding were undetectable. PSA expression was restored by complementation with the gene encoding the CMP-sialic acid transporter (CST), indicating that CST mutations were responsible for the phenotypes of the C2m cells. We characterized the CST mutations in these cells by Northern blotting and RT-PCR. C2m9 and C2m45 carried missense mutations resulting in glycine to glutamate substitutions at amino acids 217 (G217E) and 256 (G256E), respectively. C2m13, C2m39 and C2m31 had nonsense mutations that resulted in decreased CST mRNA stability, and C2m34 carried a putative splice site mutation. PSA and CD15s expression in CST-deficient Lec2 cells were partially rescued by G217E CST, but not by G256E CST, although both proteins were expressed at similar levels, and localized to the Golgi. These results indicate that the novel missense mutations isolated in this study affect CST activity.     

Two-hybrid cloning and characterization of OSH3, a yeast oxysterol-binding protein homolog

We identify Osh3p, one of seven yeast oxysterol-binding protein (OSBP) homologs, by its protein-protein interactions with a DEAD-box RNA helicase, Rok1p. The ROK1 gene was initially identified by its ability on a high-copy number plasmid to suppress the nuclear fusion defect caused by the kem1 null mutation. Our results show that OSH3 also affects nuclear fusion in a kem1-specific manner; the nuclear fusion defect of kem1 was intensified by the multicopy expression of OSH3. The Osh3p synthesis was highly induced by alpha-mating pheromone. We also found that OSH3 overexpression promoted filamentation growth of the Sigma1278b wild-type strain and suppressed the filamentation growth defect of the ste12 mutation. These results lead us to a new understanding of cellular functions of the yeast OSBPs.     

PKCdelta modulates p21WAF1/CIP1 ability to bind to Cdk2 during TNFalpha-induced apoptosis

Cyclin-dependent kinase 2 (Cdk2) activity is thought to be involved in cell death-associated chromatin condensation and other manifestations of apoptotic death. Here we show that during TNFalpha-induced apoptosis, PKCdelta is activated in a caspase-3-dependent manner and phosphorylates p21(WAF1/CIP1), a specific cyclin-dependent kinase inhibitor, on (146)Ser. This residue is located near a cyclin-binding motif (Cy2) that plays an important role in the interaction between p21(WAF1/CIP1) and Cdk2, and its phosphorylation modulates the ability of p21(WAF1/CIP1) to associate with Cdk2. The phosphorylation of p21(WAF1/CIP1) is temporally related to the activation kinetics of Cdk2 activity during the apoptosis. We propose that during TNFalpha-induced apoptosis, PKCdelta-mediated phosphorylation of p21(WAF1/CIP1) at (146)Ser attenuates the Cdk2 binding of p21(WAF1/CIP1) and thereby upregulates Cdk2 activity.     

New Record of Lycodon liuchengchaoi in Anhui

One juvenile and one adult female wolf snake(Colubridae: Lycodon) were sampled at Yixian and Fuxi, Huangshan, Anhui, China in the summer of 2011 and 2012, respectively. The two specimens were identified as Lycodon liuchengchaoi based on external morphology and molecular data. This is a new reptile record in Anhui Province. In our laboratory, four eggs were laid and three neonates were hatched successfully. This is the first record of the laying and incubation of L. liuchengchaoi eggs. The five specimens were deposited at the Museum of Huangshan University(HUM20140001) and Guangdong Entomological Institute(HB-lcfsp12613, HB-lcfsp-ch1~3).     

High-throughput evaluation of relative cell permeability between peptoids and peptides

Peptides are limited in their use as drugs due to low cell permeability and vulnerability to proteases. In contrast, peptoids are immune to enzymatic degradation and some peptoids have been shown to be relatively cell permeable. In order to facilitate future design of peptoid libraries for screening experiments, it would be useful to have a high-throughput method to estimate the cell permeability of peptoids containing different residues. In this paper, we report the strengths and limitations of a high-throughput cell-based permeability assay that registers the relative ability of steroid-conjugated peptides and peptoids to enter a cell. A comparative investigation of the physicochemical properties and side chain composition of peptoids and peptides is described to explain the observed higher cell permeability of peptoids over peptides. These data suggest that the conversion of the monomeric residues in peptides to an N-alkylglycine moiety in peptoids reduced the hydrogen-bonding potential of the molecules and is the main contributor to the observed permeability improvement.     

桦褐孔菌三萜化合物提取工艺的优选

为了提高桦褐孔菌三萜化合物的产量,对其三萜化合物的提取工艺进行了初步优选。试验以桦褐孔菌发酵菌丝体为材料,齐墩果酸为标准品,用香草醛比色法测桦褐孔菌三萜化合物含量。通过单因素实验考察了不同提取剂、提取剂用量、提取温度、提取时间对桦褐孔菌三萜化合物提取的影响。结果表明,提取的最佳工艺为：以异丙醇为提取溶剂,用量为7倍,最佳提取温度为80℃,提取时间2h,在此条件下三萜化合物占菌丝体干质量的9．03％。该工艺的三萜桦褐物的提取率高,可用于桦褐孔菌中三萜化合物的提取。     

A role for lipid rafts in immune cell signaling

Cross-linking of surface receptors in hematopoietic cells results in the enrichment of these receptors in the rafts along with other downstream signaling molecules. A possible explanation how signal is transduced through the plasma membrane has arisen from the concept of raft. From the study of cellular responses in the plasma membrane which enrich members of the Src-family tyrosine kinase, rafts can function as centers of signal transduction by forming patches. Under physiological conditions, these elements synergize to transduce successfully a signal at the plasma membrane. Rafts are suggested to be important in controlling appropriate protein interactions in hematopoietic cells, and aggregation of rafts following receptor ligation may be a general mechanism for promoting immune cell signaling.     

GalNAcβ1,3-linked paragloboside carries the epitope of a sperm maturation-related glycoprotein that is recognized by the monoclonal antibody MC121

The functional maturation of spermatozoa during epididymal transit in mammals accompanies the changes in their plasma membrane due to the binding or removal of proteins or interactions with the proteases, glycosidases and glycosyltransferases present in the epididymis. In order to study the surface changes in spermatozoa during their maturation in the epididymis, we previously established several monoclonal antibodies against the 54 kDa sialoglycoprotein of mouse cauda epididymal spermatozoa, which gradually increased the expression of antigenic determinants during epididymal transit. One of these monoclonal antibodies, MC121, reacted with mouse sperm glycoproteins on a polyvinylidene fluoride membrane after desialylation of the glycoproteins, and the treatment of the desialylated sperm glycoproteins with β-N-acetylhexosaminidase greatly decreased the expression of the antigenic determinants. In addition to reacting with mouse cauda epididymal spermatozoa, MC121 reacted with human red blood cells (hRBCs). MC121 induced agglutination of sialidase-treated hRBCs and stained hRBCs fixed with formalin vapor much more heavily than it stained hRBCs fixed with methanol. The thin layer chromatography (TLC) immunostaining of the sialidase-treated lipids of hRBCs with MC121 suggested that the epitope-bearing molecule is a glycosphingolipids (GSL), and that MC121 reacts with a pentaose-GSL. Analysis of sialidase-treated GSLs by TLC-Blot-Matrix Assisted Laser Desorption Ionization Time-of-Flight mass spectrometry (MALDI TOF MS) revealed that the GSL bound by MC121 was [HexNAc][HexNAc + Hex][Hex][Hex]-Cer. The lipid band stained with mAb TH2, which is specific for a GSL, GalNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glcβ1-ceramide. These results indicated that the epitope to which MC121 binds is present in a neolacto-series GSL, IV³GalNAcβ-nLc₄Cer² sequence.