TLR2–MyD88–NF-κB pathway is involved in tubulointerstitial inflammation caused by proteinuria

Proteinuria is an important risk factor for chronic kidney diseases (CKD). Several studies have suggested that proteinuria initiates tubulointerstitial inflammation, while the mechanisms have not been fully understood. In this study, we hypothesized whether the activation of the TLR2–MyD88–NF-κB pathway is involved in tubulointerstitial inflammation induced by proteinuria. We observed expression of TLR2, MyD88, NF-κB, as well as TNF-α and IL-6 detected by immunohistostaining, Western blotting and real-time PCR in albumin-overloaded (AO) nephropathy rats. In vitro, we observed these markers in HK-2 cells stimulated by albumin. We used TLR2 siRNA or the NF-κB inhibitor BAY 11-7082 to observe the influence of TNF-α and IL-6 expression caused by albumin overload. Finally, we studied these markers in non-IgA mesangioproliferative glomerulonephritis (MsPGN) patients with different levels of proteinuria. It was demonstrated that expression of TLR2, MyD88 and NF-κB were significantly increased in AO rats and in non-IgA MsPGN patients with high levels of proteinuria, and TNF-α and IL-6 expressions were increased after NF-κB activation. Furthermore, TNF-α and IL-6 expression was positively correlated with the level of proteinuria. Albumin-overload induced TNF-α and IL-6 secretions by the TLR2–MyD88–NF-κB pathway activation, which could be attenuated by the TLR2 siRNA or BAY 11-7082 in HK-2 cells. In summary, we demonstrated that proteinuria may exhibit an endogenous danger-associated molecular pattern (DAMP) that induces tubulointerstitial inflammation via the TLR2–MyD88–NF-κB pathway activation.     

Identification of different molecules leading to the formation of hyperanodic forms of human glucose-6-phosphate dehydrogenase

Glucose-6-phosphate dehydrogenase undergoes in vitro a decrease of its isoelectric pH in the presence of its coenzyme NADP⁺, and of either a NAD(P) glycohydrolase or an excess of its substrate, glucose-6-phosphate at acidic pHs.The mechanism of in vitro production of hyperanodic bands of glucose-6-phosphate dehydrogenase has been studied. It consists in a covalent fixation of phosphoadenosine diphosphoribose or of a degradation product of NADPH. In the case of P-ADP-Rib, the reaction is stoichiometric, one molecule of ligand being bound to one subunit of enzyme. The bond between enzyme and P-ADP-Rib was characterized as a Schiff's base.     

Glycogen synthesis by the direct or indirect pathways depends on glucose availability: in vivo studies in frog oocytes

Besides the classic direct route, frog oocytes incorporate glucosyl units into glycogen by the so-called indirect pathway. The operation of both pathways depends on glucose availability. Below 0.5 mM glucose (calculated intracellular concentration), the indirect route accounts for 90% of polysaccharide formation, while the direct pathway supports 70% of total glucose incorporation when administered glucose is above 1.5 mM. A sigmoidal curve was obtained for the direct pathway with n(H)=2.04, and half saturation was reached at 2.6 mM glucose. The curve for the indirect route presented an n(H) of 1.15 and an S(0.5) of 0.9 mM glucose.     

光/暗对玉米叶片果糖-6-磷酸激酶-2和果糖-2,6-二磷酸酯酶活力的影响

比较了照光和黑暗条件下玉米叶片果糖—6—磷酸激酶—2(PFK-2)和果糖—2,6—二磷酸酯酶(FBPase-2)的活力变化。当玉米植株从暗中转入光下后,其叶片PFK—2的活力随光照时间的延长而逐渐降低,而FBPase-2活力变化不明显;从光下转入暗后叶片PFK-2活力明显上升,FBPase-2活力仍无明显变化;其PFK-2/FBPase-2比值在光处理时下降,暗处理时上升。同时叶片中果糖—2,6—二磷酸的含量与PFK-2/FBPase-2活力比值的变化趋势一致。连续光照 20 h,PFK-2活力持续下降,表明PFK-2的光钝化现象与玉米植株的昼夜节律变化无关。     

Preparation of monoclonal antibodies against chicken liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase and their effects on enzyme activities

The effects of the monoclonal antibodies (McAbs) directed against chicken liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (6PF-2-K/Fru-2, 6-P2ase) on the structure and function of the enzyme were studied. Using chicken liver 6PF-2-K/Fru-2,5-P2asc as antigen, 7 clones of monoclonal antibodies specifically binding with the antigen were obtained. The epitopes of the antigen recognized by the 6 McAbs localized on the fructose-2,6-bisphosphatase domain of chicken liver 6PF-2-K/Fru-2, 6-P2ase, and the other (H2) are on the 6-phosphofructo-2-kinase domain. All of the 7 McAbs could activate the kinase activity of the bifunctional enzyme by twofold and had a similar effect on the bisphosphatase activity of the bifunctional enzyme which resulted in a fourfold increase of the bisphosphatase activity of the bifunctional enzyme. However, the McAbs did not affect the activity of the separated fructose-2, 6-bisphosphatase domain. The results suggested that the Fru-2, 6-P2ases in the bifunctional enzyme and     

Immunological similarity between NADH-cytochrome b5 reductase of erythrocytes and liver microsomes

In a number of animal species soluble NADH-cytochrome b₅ reductase of erythrocytes was compared with membrane-bound NADH-cytochrome b₅ reductase of liver microsomes by using an antibody to purified NADH-cytochrome b₅ reductase from rat liver microsomes. The results obtained indicated clearly that they are immunologically very similar to each other. The data with erythrocyte ghosts suggested that cytochrome b₅ and NADH-cytochrome b₅ reductase are also present in the ghost.     

Water proton relaxation as a monitor of membrane-bound manganese in spinach chloroplasts

First measurements of proton relaxation on chloroplast membranes are presented here. Experiments show that the water proton spin-lattice relaxation rate in chloroplast thylakoid membrane suspensions can be used to monitor membrane-bound manganese. The relaxation effect is reduced to 0.4 of its original value upon manganese extraction by washing with either alkaline Tris buffer or NH₂OH/EDTA solution. Large increases in the proton relaxation rate are measured in the presence of reductants such as tetraphenylboron and NH₂OH; oxidants such as potassium ferricyanide or 2,6-dichlorophenolindo-phenol lead to a decrease in this rate. These results suggest that maganese exists as a mixture of oxidation states in dark-adapted chloroplasts.     

Preliminary fabrication and characterization of electron beam melted Ti–6Al–4V customized dental implant

The current study was aimed to fabricate customized root form dental implant using additive manufacturing technique for the replacement of missing teeth. The root form dental implant was designed using Geomagic™ and Magics™, the designed implant was directly manufactured by layering technique using ARCAM A2™ electron beam melting system by employing medical grade Ti–6Al–4V alloy powder. Furthermore, the fabricated implant was characterized in terms of certain clinically important parameters such as surface microstructure, surface topography, chemical purity and internal porosity. Results confirmed that, fabrication of customized dental implants using additive rapid manufacturing technology offers an attractive method to produce extremely pure form of customized titanium dental implants, the rough and porous surface texture obtained is expected to provide better initial implant stabilization and superior osseointegration.     

Regulation of catalytic activity of S6 kinase 2 during cell cycle

Ribosomal S6 kinase 2 (S6K2) is one of the kinases regulated by the mammalian target of rapamycin (mTOR) signaling pathway. Although it has been identified as a kinase homologous to S6K1, evidence suggests that the two kinases have non-overlapping functions, and the biological function of S6K2 still remains unknown. In order to identify the cell cycle stage(s) during which S6K2 plays a role, we assessed changes in the catalytic activity of S6K2 throughout the cell cycle. Our data show that S6K2 is active throughout the cell cycle with higher activity in G2 and M phases. We also show that S6K1 activity peaks sharply during M phase. Our data suggest that S6K1 and S6K2 likely play yet-unknown roles in G2 and M phases.     

玉米果糖—6—磷酸，2—激酶／果糖—2，6—二磷酸酶基因的结构与表达分析

通过RT-PCR,结果RACE技术,得到了玉米（Zea maysL.)果糖-6-磷酸,2-激酶/果糖-2,6-二磷酸酶的全长cDNA克隆。命名为mF2KP,氨基酸序列同源性比较发现,mF2KP蛋白可以分为两个部分;C端包含高度保守的催化功能区。N端为植物中特有的多肽,将mF2KP基因中一段包含完整催化功能区的片段在大肠杆菌（Escherichia coli)中表达,融合蛋白具有果糖-6-磷酸,2-激酶/果糖-2,6-二磷酸酶活性,Northern杂交证明在种子活力不同的幼苗中,mF2KP的转录水平存在明显差异。种子活力越高,幼苗中mF2KP的转录水平越低。     

Pi和PGA对玉米叶片PFK-2/FBPase-2的作用

Fructose-6-phosphate 2-kinase and fructose 2,6-bisphosphatase have been partially purified from maize leaves by PEG fractionadon and by chromatography on TSK-DEAE ion exchanger and Blue-Sepharose 4B. Fructose-6-phosphate 2-kinase was activated by phosphate and inhibited by 3-pnosphoglycerate. Furctose 2,6-bisphosphatase was inhibited by inorganic phosphate and fructose-6-phosphate.The observed pattern of reguladon suggests that systhesis and degradation of Fru-2,6-P_2 respond to changes in the concentration of effectors. An increase in the level of glycerate-3-phosphate or dihydroxyacetonephosphate will result in a decrease in the level of Fru-2,6-P_2. Conversely a rise in Fru-6-P concentration will lead to an increase in the Fru-2, 6-P_2 concentration.     

Improvement in the expression of CYP2B6 by co-expression with molecular chaperones GroES/EL in Escherichia coli

Improvement of CYP2B6 expression was examined by co-expression with molecular chaperones GroES/EL. Although a CO-reduced difference spectrum was not detected in Escherichia coli transformed only by the CYP2B6-expressing vector, co-expression of GroES/EL resulted in high-level expression which reached over 2000 nmol P450/L. CYP2B6 was purified from the E. coli membrane with a high yield. Purified CYP2B6 showed 7-ethoxy-4-trifluoromethylcoumarin O-deethylase activity in a reconstitution system. This expression system would be useful for the production of large amounts of active CYP2B6 and for the detailed analysis of the enzyme.     

Rat liver 6-phosphofructo 2-kinase/fructose 2,6-bisphosphatase: a review of relationships between the two activities of the enzyme

Both the synthesis and the degradation of Fru-2,6-P2 are catalyzed by a single enzyme protein; ie, the enzyme is bifunctional. This protein, which we have designated 6-phosphofructo 2-kinase/fructose 2,6-bisphosphatase is an important enzyme in the regulation of hepatic carbohydrate metabolism since its activity determines the steady-state concentration of fructose 2,6-P2, an activator of 6-phosphofructo 1-kinase and an inhibitor of fructose 1,6-bisphosphatase. Regulation of the bifunctional enzyme in intact cells is a complex function of both covalent modification via phosphorylation/dephosphorylation and the influence of substrates and low molecular weight effectors. Recent evidence suggests that both reactions may proceed by two-step transfer mechanisms with different phosphoenzyme intermediates. The enzyme catalyzes exchange reactions between ADP and ATP and between fructose 6-P and fructose 2,6-P2. A labeled phosphoenzyme is formed rapidly during incubation with [2-32P]Fru-2,6-P2. The labeled residue has been identified as 3-phosphohistidine. However, it was not possible to demonstrate significant labeling of the enzyme directly from [gamma-32P]ATP. These results can be most readily explained in terms of two catalytic sites, a kinase site whose phosphorylation by ATP is negligible (or whose E-P is labile) and a fructose 2,6-bisphosphatase site which is readily phosphorylated by fructose 2,6-P2. Additional evidence in support of two active sites include: limited proteolysis with thermolysin results in loss of 6-phosphofructo 2-kinase activity and activation of fructose 2,6-bisphosphatase, mixed function oxidation results in inactivation of the 6-phosphofructo 2-kinase but no affect on the fructose 2,6-bisphosphatase, N-ethylmaleimide treatment also inactivates the kinase but does not affect the bisphosphatase, and p-chloromercuribenzoate immediately inactivates the fructose 2,6-bisphosphatase but not the 6-phosphofructo 2-kinase. Our findings indicate that the bifunctional enzyme is a rather complicated enzyme; a dimer, probably with two catalytic sites reacting with sugar phosphate, and with an unknown number of regulatory sites for most of its substrates and products. Three enzymes from Escherichia coli, isocitric dehydrogenase kinase/phosphatase, glutamine-synthetase adenylyltransferase, and the uridylyltransferase for the regulatory protein PII in the glutamine synthetase cascade system also catalyze opposing reactions probably at two discrete sites. All four enzymes are important in the regulation of metabolism and may represent a distinct class of regulatory enzymes.     

Nature and position of functional group on thiopurine substrates influence activity of xanthine oxidase — Enzymatic reaction pathways of 6-mercaptopurine and 2-mercaptopurine are different

Xanthine oxidase-catalyzed hydroxylation reactions of the anticancer drug 6-mercaptopurine (6-MP) and its analog 2-mercaptopurine (2-MP) as well as 6-thioxanthine (6-TX) and 2-thioxanthine (2-TX) have been studied using UV-spectroscopy, high pressure liquid chromatography, photodiode array, and liquid chromatography-based mass spectral analysis. It is shown that 6-MP and 2-MP are oxidatively hydroxylated through different pathways. Enzymatic hydroxylation of 6-MP forms 6-thiouric acid in two steps involving 6-TX as the intermediate, whereas 2-MP is converted to 8-hydroxy-2-mercaptopurine as the expected end product in one step. Surprisingly, in contrast to the other thiopurines, enzymatic hydroxylation of 2-MP showed a unique hyperchromic effect at 264 nm as the reaction proceeded. However, when 2-TX is used as the substrate, it is hydroxylated to 2-thiouric acid. The enzymatic hydroxylation of 2-MP is considerably faster than that of 6-MP, while 6-TX and 2-TX show similar rates under identical reaction conditions. The reason why 2-MP is a better substrate than 6-MP and how the chemical nature and position of the functional groups present on the thiopurine substrates influence xanthine oxidase activity are discussed.     

Effects of 1-methyl cyclohexane carboxylic acid (CCA) on cellular energetics in neuroblastoma cells

The level of cellular energetics has been estimated in neuroblastoma cells under different culture conditions. The cellular accumulation of isomerase-2-deoxy [¹⁴C]D-glucose-6-phosphate was taken as reflecting glucose utilization. A considerably higher amount of radioactivity — 2.5 to 4 times — is found in CCA treated cells, as compared to other types of cultures, corresponding to a higher rate of deoxyglucose penetration and utilization.     

Discovery of (E)-5-(benzylideneamino)-1H-benzo[d]imidazol-2(3H)-one derivatives as inhibitors for PTK6

A lead compound 1, which inhibits the catalytic activity of PTK6, was selected from a chemical library. Derivatives of compound 1 were synthesized and analyzed for inhibitory activity against PTK6 in vitro and at the cellular level. Selected compounds were analyzed for cytotoxicity in human foreskin fibroblasts using MTT assays and for selectivity towards PTK members in HEK 293 cells. Compounds 20 (in vitro IC₅₀ = 0.12 μM) and 21 (in vitro IC₅₀ = 0.52 μM) showed little cytotoxicity, excellent inhibition of PTK6 in vitro and at the cellular level, and selectivity for PTK6. Compounds 20 and 21 inhibited phosphorylation of specific PTK6 substrates in HEK293 cells. Thus, we have identified novel PTK6 inhibitors that may be used as treatments for PTK6-positive carcinomas, including breast cancer.     

Evidence for an inducible glucose transport system in Kluyveromyces lactis

To find the cause of delayed glucose oxidation in succinate-grown Kluyveromyces lactis, glucose transport was studied in glucose- and in succinate-grown cells. The initial rate of 2-deoxyglucose (2-dGlc) accumulation, as well as the appearance of 2-deoxyglucose 6-phosphate, was higher in the glucose-grown cells. In both cell types, 2-dGlc was apparently transported in the free form to be phosphorylated intracellularly . In glucose-grown cells the level of free 2-dGlc in the pool was always less than the external concentration. Exchange transport in starved, poisoned cells loaded with unlabeled 2-dGlc was 140-fold greater in glucose- than in succinate-grown cells, probably because of the presence of an inducible transport component. The development of the increased rate of transport in a succinate-grown uracil-requiring auxotroph after transfer to glucose depends on the presence of uracil.