Membrane fouling in sterile filtration of recombinant human growth hormone

The most troublesome problem encountered during the sterile filtration of protein solutions is membrane fouling. This article presents our study on sterile filtration of a model protein, recombinant human growth hormone (rhGH). Scanning electron microscopy (SEM) analysis shows that 0.22-mum membranes, when used to filter the mannitol-formulated protein solution under a 0.35-bar transmembrane pressure, were plugged to a great extent. When zinc ions were added to induce aggregates, the fouling tendency of rhGH solutions increased with increasing amount and size of the aggregates, indicating that the aggregates present before filtration might be responsible for membrane fouling. However, repeated filtration of the same solution using a fresh filter each time cannot reduce membrane fouling, and all filtrates contain the same trace amount of hGH particulates as the prefiltered solution. Particulate size was determined to be between 0.03 and 0.15 mum by dynamic light scattering. Also, in view of the fact that protein formulations significantly affected the tendency of fouling with the same preexisting aggregates, it is likely that fouling was more attributed to the aggregation taking place in the filter pores during filtration (secondary aggregation) than to the aggregates present before filtration. Adding a surfactant to or increasing the pH of the protein solution improves the filtration, whereas increasing ionic strength slows down the filtration. This result suggests that the balance of the protein's interaction and electrostatic repulsion plays an important role in the protein's fouling tendency. Many factors might change the microenvironment in the pores and disturb this balance. Those considerations and the aggregation tendency of rhGH in the filter pores will be discussed in detail separately. (c) 1996 John Wiley & Sons, Inc.     

Developmentally regulated sphingolipid synthesis in African trypanosomes

Sphingolipids are essential components of eukaryotic membranes, and many unicellular eukaryotes, including kinetoplastid protozoa, are thought to synthesize exclusively inositol phosphorylceramide (IPC). Here we characterize sphingolipids from Trypanosoma brucei, and a trypanosome sphingolipid synthase gene family (TbSLS1-4) that is orthologous to Leishmania IPC synthase. Procyclic trypanosomes contain IPC, but also sphingomyelin, while surprisingly bloodstream-stage parasites contain sphingomyelin and ethanolamine phosphorylceramide (EPC), but no detectable IPC. In vivo fluorescent ceramide labelling confirmed stage-specific biosynthesis of both sphingomyelin and IPC. Expression of TbSLS4 in Leishmania resulted in production of sphingomyelin and EPC suggesting that the TbSLS gene family has bi-functional synthase activity. RNAi silencing of TbSLS1-4 in bloodstream trypanosomes led to rapid growth arrest and eventual cell death. Ceramide levels were increased more than threefold by silencing suggesting a toxic downstream effect mediated by this potent intracellular messenger. Topology predictions support a revised six-transmembrane domain model for the kinetoplastid sphingolipid synthases consistent with the proposed mammalian sphingomyelin synthase structure. This work reveals novel diversity and regulation in sphingolipid metabolism in this important group of human parasites.     

CCL5/CCR5 axis promotes the motility of human oral cancer cells

CCL5 (previously called RANTES) is in the CC‐chemokine family and plays a crucial role in the migration and metastasis of human cancer cells. On the other hand, the effect of CCL5 is mediated via CCR receptor. RT‐PCR and flow cytometry studies demonstrated CCR5 but not CCR1 and CCR3 mRNA in oral cancer cell lines, especially higher in those with high invasiveness (SCC4) as compared with lower levels in HSC3 cells and SCC9 cells. Stimulation of oral cancer cells with CCL5 directly increased the migration and metalloproteinase‐9 (MMP‐9) production. MMP‐9 small interfering RNA inhibited the CCL5‐induced MMP‐9 expression and thereby significantly inhibited the CCL5‐induced cell migration. Activations of phospholipase C (PLC), protein kinase Cδ (PKCδ), and NF‐κB pathways after CCL5 treatment was demonstrated, and CCL5‐induced expression of MMP‐9 and migration activity was inhibited by the specific inhibitor of PLC, PKCδ, and NF‐κB cascades. In addition, migration‐prone sublines demonstrate that cells with increasing migration ability had more expression of MMP‐9, CCL5, and CCR5. Taken together, these results indicate that CCL5/CCR5 axis enhanced migration of oral cancer cells through the increase of MMP‐9 production. J. Cell. Physiol. 220: 418–426, 2009. © 2009 Wiley‐Liss, Inc.     

N-Acetyl-d-glucosamine 2-epimerase from Anabaena sp. CH1 contains a novel ATP-binding site required for catalytic activity

ATP is required as a structural activator for the reversible epimerization of N-acetyl-d-glucosamine to N-acetyl-d-mannosamine by N-acetyl-d-glucosamine 2-epimerase (AGE); however, the ATP-binding site on AGE has not been clearly identified. This study aimed to investigate the specific region of Anabaena sp. CH1 AGE (bAGE) that is required for ATP binding. In the absence of ATP, tryptic digest of bAGE resulted in the production of 2 segments of 17 and 26 kDa, while in the presence of 1 mM ATP, the enzyme was resistant to trypsin. ADP also displayed protective effects against trypsin digestion. A trypsin-mediated ATP-footprinting assay identified a deviant ATP-protected region, 156-GKYTK-160, which is located within the flexible loop of bAGE. Site-directed mutagenesis of residues in the loop region was performed, and both K151A and K160A variants greatly decreased the enzymatic activity as well as the ATP-binding ability of bAGE, indicating that residues K151 and K160 may be critical for ATP binding. This study demonstrated that the ATP-binding site (151-KDNPKGKYTK-160) of bAGE was a novel rather than a classical Walker motif A. This is the first ATP-binding site reported for AGEs.     

Cloning and Expression of a Schwanniomyces occidentalis alpha-Amylase Gene in Saccharomyces cerevisiae

An alpha-amylase gene (AMY) was cloned from Schwanniomyces occidentalis CCRC 21164 into Saccharomyces cerevisiae AH22 by inserting Sau3AI-generated DNA fragments into the BamHI site of YEp16. The 5-kilobase insert was shown to direct the synthesis of alpha-amylase. After subclones containing various lengths of restricted fragments were screened, a 3.4-kilobase fragment of the donor strain DNA was found to be sufficient for alpha-amylase synthesis. The concentration of alpha-amylase in culture broth produced by the S. cerevisiae transformants was about 1.5 times higher than that of the gene donor strain. The secreted alpha-amylase was shown to be indistinguishable from that of Schwanniomyces occidentalis on the basis of molecular weight and enzyme properties.     

Integration of tomato reproductive developmental landmarks and expression profiles, and the effect of SUN on fruit shape

Background  

Universally accepted landmark stages are necessary to highlight key events in plant reproductive development and to facilitate comparisons among species. Domestication and selection of tomato resulted in many varieties that differ in fruit shape and size. This diversity is useful to unravel underlying molecular and developmental mechanisms that control organ morphology and patterning. The tomato fruit shape gene SUN controls fruit elongation. The most dramatic effect of SUN on fruit shape occurs after pollination and fertilization although a detailed investigation into the timing of the fruit shape change as well as gene expression profiles during critical developmental stages has not been conducted.     

Deletion of peptide amidation enzymatic activity leads to edema and embryonic lethality in the mouse

Peptidylglycine alpha-amidating monooxygenase (PAM) catalyzes the COOH-terminal amidation of peptide hormones. We previously had found high expression of PAM in several regions of the developing rodent. To determine the function of PAM during mouse embryogenesis, we produced a null mutant of the PAM gene. Homozygous mutants die in utero between e14.5 and e15.5 with severe edema that is likely due to cardiovascular deficits. These defects include thinning of the aorta and carotid arteries and are very similar to those of the recently characterized adrenomedullin (AM) gene KO despite the presence of elevated immunoreactive AM in PAM KO embryos. No peptide amidation activity was detected in PAM mutant embryos, and there was no moderation of the AM-like phenotype that could be expected if any alternative peptide amidation mechanism exists in the mouse. Despite the proposed contribution of amidated peptides to neuronal cell proliferation, no alteration in neuroblast proliferation was observed in homozygous mutant embryos prior to lethality. Mice heterozygous for the mutant PAM allele develop normally and express wildtype levels of several amidated peptides despite having one half the wildtype levels of PAM activity and PAM protein. Nonetheless, both an increase in adiposity and a mild glucose intolerance developed in aged (>10 months) heterozygous mice compared to littermate controls. Ablation of PAM thus demonstrates an essential function for this gene during mouse development, while alterations in PAM activity in the adult may underlie more subtle physiologic effects.     

Sequence features involved in the mechanism of 3' splice junction wobbling

Background  

Alternative splicing is an important mechanism mediating the diversified functions of genes in multicellular organisms, and such event occurs in around 40-60% of human genes. Recently, a new splice-junction wobbling mechanism was proposed that subtle modifications exist in mRNA maturation by alternatively choosing at 5'- GTNGT and 3'- NAGNAG, which created single amino acid insertion and deletion isoforms.