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1.
Esterase G (EstG) from dibutyl phthalate (DBP)-degrading Sphingobium sp. SM42 was immobilized on amine-functionalized supports through aldehyde tag technology. Two different sulfatase motif tags, either LCTPSR (cysteine-type) or MSAPAR (serine-type), each of which is recognized by a specific formylglycine generating enzyme (FGE), were fused to the C-terminus of EstG. The cysteine-specific FGE was derived from Pseudomonas putida KT2440 while Klebsiella sp. SLS5 provided serine-specific FGE. The EstG with serine-type aldehyde tag showed a greater immobilization yield and higher specific activity by 4.8-fold and 1.8-fold, respectively. The immobilized EstG retained over 90% of its original activity after seven cycles of usage, and exhibited significantly improved thermostability by retaining 66% activity after 1 h incubation at 60 °C. Additionally, nearly 100% and over 30% of the DBP in 10 mM and 100 mM solutions, respectively, was degraded by the immobilized EstG within 18 h.  相似文献   
2.
Palmitic acid (PA) is associated with higher blood concentrations of medium-chain acylcarnitines (MCACs), and we hypothesized that PA may inhibit progression of FA β-oxidation. Using a cross-over design, 17 adults were fed high PA (HPA) and low PA/high oleic acid (HOA) diets, each for 3 weeks. The [1-13C]PA and [13-13C]PA tracers were administered with food in random order with each diet, and we assessed PA oxidation (PA OX) and serum AC concentration to determine whether a higher PA intake promoted incomplete PA OX. Dietary PA was completely oxidized during the HOA diet, but only about 40% was oxidized during the HPA diet. The [13-13C]PA/[1-13C]PA ratio of PA OX had an approximate value of 1.0 for either diet, but the ratio of the serum concentrations of MCACs to long-chain ACs (LCACs) was significantly higher during the HPA diet. Thus, direct measurement of PA OX did not confirm that the HPA diet caused incomplete PA OX, despite the modest, but statistically significant, increase in the ratio of MCACs to LCACs in blood.  相似文献   
3.
High glucose concentrations due to diabetes increase apoptosis of vascular pericytes, impairing vascular regulation and weakening vessels, especially in brain and retina. We sought to determine whether vitamin C, or ascorbic acid, could prevent such high glucose-induced increases in pericyte apoptosis. Culture of human microvascular brain pericytes at 25 mM compared to 5 mM glucose increased apoptosis measured as the appearance of cleaved caspase 3. Loading the cells with ascorbate during culture decreased apoptosis, both at 5 and 25 mM glucose. High glucose-induced apoptosis was due largely to activation of the receptor for advanced glycation end products (RAGE), since it was prevented by specific RAGE inhibition. Culture of pericytes for 24 h with RAGE agonists also increased apoptosis, which was completely prevented by inclusion of 100 μM ascorbate. Ascorbate also prevented RAGE agonist-induced apoptosis measured as annexin V binding in human retinal pericytes, a cell type with relevance to diabetic retinopathy. RAGE agonists decreased intracellular ascorbate and GSH in brain pericytes. Despite this evidence of increased oxidative stress, ascorbate prevention of RAGE-induced apoptosis was not mimicked by several antioxidants. These results show that ascorbate prevents pericyte apoptosis due RAGE activation. Although RAGE activation decreases intracellular ascorbate and GSH, the prevention of apoptosis by ascorbate may involve effects beyond its function as an antioxidant.  相似文献   
4.
5.
Sterol O-acyltransferase 2 (SOAT2), also known as ACAT2, is the major cholesterol esterifying enzyme in the liver and small intestine (SI). Esterified cholesterol (EC) carried in certain classes of plasma lipoproteins is hydrolyzed by lysosomal acid lipase (LAL) when they are cleared from the circulation. Loss-of-function mutations in LIPA, the gene that encodes LAL, result in Wolman disease (WD) or cholesteryl ester storage disease (CESD). Hepatomegaly and a massive increase in tissue EC levels are hallmark features of both disorders. While these conditions can be corrected with enzyme replacement therapy, the question arose as to what effect the loss of SOAT2 function might have on tissue EC sequestration in LAL-deficient mice. When weaned at 21 days, Lal/:Soat2+/+ mice had a whole liver cholesterol content (mg/organ) of 24.7 mg vs 1.9 mg in Lal+/+:Soat2+/+ littermates, with almost all the excess sterol being esterified. Over the next 31 days, liver cholesterol content in the Lal/:Soat2+/+ mice increased to 145 ± 2 mg but to only 29 ± 2 mg in their Lal/:Soat2/ littermates. The level of EC accumulation in the SI of the Lal/:Soat2/ mice was also much less than in their Lal/:Soat2+/+ littermates. In addition, there was a >70% reduction in plasma transaminase activities in the Lal/:Soat2/ mice. These studies illustrate how the severity of disease in a mouse model for CESD can be substantially ameliorated by elimination of SOAT2 function.  相似文献   
6.
Signal peptides are short peptides located at the N-terminus of secreted proteins. They characteristically have three domains; a basic region at the N-terminus (n-region), a central hydrophobic core (h-region) and a carboxy-terminal cleavage region (c-region). Although hundreds of different signal peptides have been identified, it has not been completely understood how their features enable signal peptides to influence protein expression. Antibody-derived signal peptides are often used to prepare recombinant antibodies expressed by eukaryotic cells, especially Chinese hamster ovary (CHO) cells. However, when prokaryotic Escherichia coli (E. coli) are utilized in drug discovery processes, such as for phage display selection or antibody humanization, signal peptides have been selected separately due to the differences in the expression systems between the species. In this study, we successfully established a signal peptide that enables a functional antibody to be expressed in both prokaryotic and eukaryotic cells by focusing on the importance of having an Ala residue in the c-region of the signal sequence. We found that changing Ser to Ala at only two positions significantly augmented the anti-HER2 antigen binding fragment (Fab) expression in E. coli. In addition, this altered signal peptide also retained the ability to express functional anti-HER2 antibody in CHO cells. Taken together, the present findings indicate that the signal peptide can promote functional antibody expression in both prokaryotic E. coli and eukaryotic CHO cells. This finding will contribute to the understanding of signal peptides and accelerate therapeutic antibody research.  相似文献   
7.
Unique species of ceramide (Cer) with very-long-chain polyunsaturated fatty acid (VLCPUFA), mainly 28–32 carbon atoms, 4–5 double bonds, in nonhydroxy and 2-hydroxy forms (n-V Cer and h-V Cer, respectively), are generated in rat spermatozoa from the corresponding sphingomyelins during the acrosomal reaction. The aim of this study was to determine the properties of these sperm-distinctive ceramides in Langmuir monolayers. Individual Cer species were isolated by HPLC and subjected to analysis of surface pressure, surface potential, and Brewster angle microscopy (BAM) as a function of molecular packing. In comparison with known species of Cer, n-V Cer and h-V Cer species showed much larger mean molecular areas and increased molecular dipole moments in liquid expanded phases, which suggest bending and partial hydration of the double bonded portion of the VLCPUFA. The presence of the 2-hydoxyl group induced a closer molecular packing in h-V Cer than in their chain-matched n-V Cer. In addition, all these Cer species showed liquid-expanded to liquid-condensed transitions at room temperature. Existence of domain segregation was confirmed by BAM. Additionally, thermodynamic analysis suggests a phase transition close to the physiological temperature for VLCPUFA-Cers if organized as bulk dispersions.  相似文献   
8.
The insulin receptor (IR) binds insulin and plays important roles in glucose homeostasis by regulating the tyrosine kinase activity at its C-terminus. Its transmembrane domain (TMD) is shown to be important for transferring conformational changes induced by insulin across the cell membrane to regulate kinase activity. In this study, a construct IR940–988 containing the TMD was expressed and purified for structural studies. Its solution structure in dodecylphosphocholine (DPC) micelles was determined. The sequence containing residues L962 to Y976 of the TMD of the IR in micelles adopts a well-defined helical structure with a kink formed by glycine and proline residues present at its N-terminus, which might be important for its function. Paramagnetic relaxation enhancement (PRE) and relaxation experimental results suggest that residues following the TMD are flexible and expose to aqueous solution. Although purified IR940–988 in micelles existed mainly as a monomeric form verified by gel filtration and relaxation analysis, cross-linking study suggests that it may form a dimer or oligomers under micelle conditions.  相似文献   
9.
Dystrophin (DYS) is a filamentous protein that connects the cytoskeleton and the extracellular matrix via the sarcolemma, conferring resistance to muscular cells. In this study, interactions between the DYS R16–21 fragment and lipids were examined using Langmuir films made of anionic and zwitterionic lipids. The film fluidity was modified by the addition of 15% cholesterol. Whatever the lipid mixture examined, at low surface pressure (20 mN/m) few differences appeared on the protein insertion and the presence of cholesterol did not affect the protein/lipid interactions. At high surface pressure (30 mN/m), the protein insertion was very low and occurred only in zwitterionic films in the liquid-expanded phase. In anionic films, electrostatic interactions prevented the protein insertion outright, and caused accumulation of the protein on the hydrophilic part of the monolayer. Addition of cholesterol to both lipid mixtures drastically modified the protein–lipid interactions: the DYS R16–21 insertion increased and its organization in the monolayer appeared to be more homogeneous. The presence of accessible cholesterol recognition amino-acid consensus sequences in this fragment may enhance the protein/membrane binding at physiological lateral pressure. These results suggest that the anchorage of dystrophin to the membrane in vivo may be stabilized by cholesterol-rich nano-domains in the inner leaflet of sarcolemma.  相似文献   
10.
We have analyzed the cell wall of the radio-resistant bacterium Deinococcus radiodurans. Unexpectedly, the bacterial envelope appears to be organized in different complexes of high molecular weight. Each complex is composed of several proteins, most of which are coded by genes of unknown function and the majority are constituents of the inner/outer membrane system. One of the most abundant complexes is constituted by the gene DR_0774. This protein is a type of secretin which is a known subunit of the homo-oligomeric channel that represents the main bulk of the type IV piliation family. Finally, a minor component of the pink envelope consists of several inner-membrane proteins. The implications of these findings are discussed.  相似文献   
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