The use of regenerable, affinity ligand-based surfaces for immunosensor applications.

The regeneration of antibody-binding surfaces is of major importance for re-usable sensor formats such as required for direct 'real-time' biosensing technologies and is often difficult to achieve. Antibodies commonly bind the antigen with high avidity and may themselves be sensitive to regeneration conditions. The interaction of polyclonal anti-chlorpyriphos antibody with an immobilised chlorpyriphos-ovalbumin (chlor-oval) conjugate and the interaction of soluble recombinant CD4 with covalently immobilised anti-CD4 IgG are presented in order to highlight these difficulties. Affinity-capture is suggested as an alternative format as it facilitates surface regeneration, directed immobilisation and the attainment of interaction progress curves that conform to the ideal pseudo-first-order kinetic interaction model. Protein A, protein G and polyclonal anti-mouse Fe-coated surfaces were used to observe the interaction of captured anti-GST monoclonal antibody with glutathione-s-transferase (GST). It was shown that a protein A affinity-capture surface produced ideal interaction progress curves while both protein G and polyclonal anti-mouse Fe resulted in systemic deviations.     

Erythrocyte antioxidant enzymes in toxicological evaluation of commonly used organophosphate pesticides

Erythrocytes are excellent models for the study of interactions of xenobiotics with biomembranes. Present work is designed to study the in vitro effects of some organophosphates (ethion, chlorpyrifos, dimethoate and monocrotophos) on rat erythrocytes. Treatment of erythrocytes with organophosphates resulted in decreased erythrocyte glucose-6-phosphate dehydrogenase (G-6-PD) activity, whereas activities of glutathione-s-transferase (GST) and glutathione reductase (GR) were increased. Reduced Glutathione (GSH) content of RBCs was decreased after treatment with the pesticides. Increased activities of GST and GR were due to induction of natural defense mechanism of erythrocytes against the toxicity of the pesticides. Membrane bound enzymes like acetylcholinesterase (AChE), Na(+)-K(+)-ATPase and Ca(2+)-ATPase were also inhibited. Altered activities of these enzymes along with decreased GSH content indicate increased oxidative stress in erythrocytes after treatment with organophosphates.     

Induction and signaling of an apoptotic response typified by DNA laddering in the defense response of oats to infection and elicitors

Cells in the primary leaves of oats displayed internucleosomal DNA cleavage in response to incompatible crown rust infection. DNA laddering also was evident in leaves treated with calcium ionophore A23187, nonspecific elicitors such as chitin and chitosan oligomers, and victorin, which functions as a specific elicitor in Pc-2/Vb containing oat leaves. The nuclei in a victorin-treated susceptible oat line were positive for the TUNEL assay. These elicitors clearly induced a 28-kDa nuclease (p28) in addition to three constitutive nucleases of 33, 24, and 22 kDa. Activation of p28 preceded the appearance of DNA laddering and possibly was mediated by de novo synthesis and/or cysteine protease activity. Pharmacological studies showed that the induction of DNA laddering was associated with oxidative stress, Ca2+ influx, and serine and cysteine proteases. Protein kinase and calmodulin activities did not seem to be involved in the induction of DNA laddering by victorin, whereas kinase-mediated signals were involved in DNA laddering induced by A23187. Protein kinase, calmodulin, G-protein activities, and Ca2+ influx, however, are involved in phytoalexin production. Our results imply that p28 is a possible nuclease candidate responsible for the induction of DNA laddering. The results also demonstrated that the mediators involved in the induction of apoptosis depended on the type of stimuli, whereas p28 and serine and cysteine proteases commonly are associated with each elicitor-induced apoptosis.     

Soluble and Insoluble Protein Patterns during Induction of Freezing Tolerance in Black Locust Seedlings

Protein metabolism plays a major role in the development of freezing tolerance in plants. Soluble and insoluble protein concentrations were followed during induction of freezing tolerance in black locust (Robinia pseudoacacia L.) stem tissues. Soluble proteins were fractionated using two-dimensional polyacrylamide gel electrophoresis and examined for the presence of glycoprotein fractions. Soluble protein concentration remained relatively constant during early stages of induction of freezing tolerance but increased significantly during later stages, while insoluble protein concentration remained relatively constant throughout induction. A new soluble protein component appeared during later stages of induction and was identified as a glycoprotein. Some glycoproteins are known to have a high water-binding capacity, which could play a role in intracellular resistance to ice formation during development of freezing tolerance.     

Distinct Roles of Protein Phosphatase 1 Bound on Neurabin and Spinophilin and Its Regulation in AMPA Receptor Trafficking and LTD Induction

Protein phosphatase-1 (PP1) constrains learning and memory formation in part through its effects on the induction threshold of long-term potentiation (LTP) and depression (LTD). LTD induction requires both the enzymatic activity of PP1 and its proper anchoring to synaptic spines. We have shown previously that neurabin, a major synaptic scaffolding protein, targets PP1 to synapses for LTD induction. Here, we show that PP1 bound on spinophilin, a close homolog of neurabin and another major synaptic PP1 anchoring protein, does not play a role in LTD induction, which suggests that neurabin plays a privileged role in nanodomain targeting of PP1 in LTD induction. We found that protein kinase A can significantly weaken the neurabin-PP1 interaction in neurons via phosphorylation of neurabin at serine 461, a phosphorylation site adjacent to the PP1-binding motif that is not conserved in spinophilin. Finally, we found that a neurabin mutation (S461E), which mimics phosphorylation, blocked AMPA receptor endocytosis and LTD induction. The results indicate the critical importance of nanodomain targeting of PP1 within synaptic spines and its regulation in LTD induction.     

Glycyrrhizin exhibits potential chemopreventive activity on 12-O-tetradecanoyl phorbol-13-acetate-induced cutaneous oxidative stress and tumor promotion in Swiss albino mice

Glycyrrhizin and its aglycone, glycyrrhetic acid has been found useful for various therapeutic purposes. Glycyrrhizin has been shown to possess many physiological functions like anti-inflammatory activity, detoxification and inhibition of carcinogenic promoters. 12-O-Tetradecanoyl phorbol-13-acetate (TPA), a well-known phorbal ester is known for its tumor promotion activity. The induction of inflammation in skin mediated by TPA is believed to be governed by cyclooxygenase (COX), lipoxygenase and ornithine decarboxylase (ODC). These markers of inflammatory responses are important for skin tumor promotion. In our present study, we studied the chemopreventive effect of glycyrrhizin on TPA (20 nmol/0.2 mL acetone/animal, topically)-induced oxidative stress and hyperproliferation markers in skin. TPA enhanced lipid peroxidation with reduction in the level of catalase, glutathione, glutathione peroxidase, glutathione reductase and glutathione-s-transferase. TPA treatment also enhanced ODC activity and [3H] thymidine incorporation into cutaneous DNA. Prophylactic treatment of mice with glycyrrhizin (2.0 & 4.0 mg/0.2 mL acetone/animal, topically) resulted in a significant decrease in cutaneous microsomal lipid peroxidation (P < 0.001) and recovery of cutaneous glutathione content (P < 0.001) and its dependent enzymes. A significant inhibition in ODC activity and DNA synthesis (P < 0.001) was also observed. Thus, the results demonstrate that pretreatment with glycyrrhizin is protective against TPA-induced oxidative stress and tumor promotion in Swiss albino mice.     

PP2A通过Akt/Skp2通路调控p27~(Kip1)的表达并抑制肺癌细胞的致瘤能力

蛋白磷酸酶2A(PP2A)是由36 k Da的催化亚基C(PP2Ac)和65 k Da的结构亚基A(PP2Aα/β)一起组成PP2A的核心酶,并且和各种不同的调节亚基B形成具有不同功能的PP2A全酶复合体。在细胞中PP2A发挥着重要作用,特别是在抑制肿瘤的形成当中,编码PP2Aα/β基因的突变将导致肿瘤的形成和其他疾病。当非小细胞肺癌细胞H1299中过表达PP2A-Aα时,细胞生长被抑制,细胞周期停留在G0/G1期,致瘤能力也同时被抑制。进一步研究证明当PP2A-Aα过表达时,Akt被去磷酸化失活使Skp2的表达下调,从而导致细胞周期抑制因子p27kip1的表达上调。肿瘤细胞软琼脂克隆形成实验的结果表明过表达PP2A-Aα之后H1299细胞的锚定非依赖性生长能力明显的降低,形成的克隆细胞团也较小,这些结果和裸鼠成瘤实验的结果是一致的。     

Overproduction and purification of protein P6 of Bacillus subtilis phage phi 29: role in the initiation of DNA replication.

A phi 29 DNA fragment containing gene 6, required for DNA replication, has been cloned in plasmid pPLc28 under the control of the PL promoter of phage lambda. A polypeptide with an electrophoretic mobility close to that of p6 was labelled with 35S-methionine after heat induction. This protein, representing about 4% of the total E. coli protein after 1 h of induction, was obtained in a highly purified form. The protein was characterized as p6 by amino acid analysis and NH2-and COOH-terminal sequence determination. Protein p6 has an apparent molecular weight of 23,600, suggesting that the native form of the protein is a dimer. The purified protein p6 stimulated the protein-primed initiation of phi 29 DNA replication when added to purified proteins p2 (phi 29-coded DNA polymerase) and p3 (terminal protein).     

Impact of chromium on lipoperoxidative processes and subsequent operation of the glutathione cycle in rat renal system.

Oral administration of K2Cr2O7 to male albino rats at an acute dose of 1500 mg/kg body wt/day for 3 days brought about sharp decrease in the activities of glucose-6-phosphate dehydrogenase and glutathione reductase of kidney epithelial cells. The scavenging system of kidney epithelium is also affected as evident by the highly significant fall in the activities of glutathione peroxidase, superoxide dismutase and catalase which ultimately leads to the increase in lipid peroxidation value in kidney cortical homogenate. However, glutathione-s-transferase activity in cytosol and glutathione and total thiol content in cortical homogenate were not altered. Chronic oral administration of K2Cr2O7 (300 mg/kg body wt/day) for 30 days to rats lead to elevation in the activities of glutathione peroxidase, glutathione reductase, glutathione-s-transferase, superoxide dismutase and catalase with no change in glucose-6-phosphate dehydrogenase activity in epithelial cells. This might lead to the increase in glutathione and total thiol status and decrease in lipid peroxidation value in whole homogenate system.     

A yeast 2-hybrid analysis of human GTP cyclohydrolase I protein interactions

The yeast 2-hybrid system was used to identify protein domains involved in the oligomerization of human guanosine 5'-triphosphate (GTP) Cyclohydrolase I (GCH1) and the interaction of GCH1 with its regulatory partner, GCH1 feedback regulatory protein (GFRP). When interpreted within the structural framework derived from crystallography, our results indicate that the GCH1 N-terminal alpha-helices are not the only domains involved in the formation of dimers from monomers and also suggest an important role for the C-terminal alpha-helix in the assembly of dimers to form decamers. Moreover, a previously unknown role of the extended N-terminal alpha-helix in the interaction of GCH1 and GFRP was revealed. To discover novel GCH1 protein binding partners, we used the yeast 2-hybrid system to screen a human brain library with GCH1 N-terminal amino acids 1-96 as prey. This protruding extension of GCH1 contains two canonical Type-I Src homology-3 (SH3) ligand domains located within amino acids 1-42. Our screen yielded seven unique clones that were subsequently shown to require amino acids 1-42 for binding to GCH1. The interaction of one of these clones, Activator of Heat Shock 90 kDa Protein (Aha1), with GCH1 was validated by glutathione-s-transferase (GST) pull-down assay. Although the physiological relevance of the Aha1-GCH1 interaction requires further study, Aha1 may recruit GCH1 into the endothelial nitric oxide synthase/heat shock protein (eNOS/Hsp90) complex to support changes in endothelial nitric oxide production through the local synthesis of BH4.     

Protein and Ribonucleic Acid Synthesis During the Diploid Life Cycle of Allomyces arbuscula

The diploid life cycle of Allomyces arbuscula may be divided into four parts: spore induction, germination, vegetative growth, and mitosporangium formation. Spore induction, germination, and mitosporangium formation are insensitive to inhibition of actinomycin D, probably indicating that stable, pre-existing messenger ribonucleic acid (RNA) is responsible for these developmental events. Protein synthesis is necessary during the entire life cycle except for cyst formation. A system for obtaining synchronous germination of mitospores is described. During germination there is a characteristic increase in the rate of synthesis of RNA and protein although none of the other morphogenetic changes occurring during the life cycle are necessarily accompanied by an appreciable change in the rate of macromolecular synthesis.     

Exonuclease-1 deletion impairs DNA damage signaling and prolongs lifespan of telomere-dysfunctional mice

Exonuclease-1 (EXO1) mediates checkpoint induction in response to telomere dysfunction in yeast, but it is unknown whether EXO1 has similar functions in mammalian cells. Here we show that deletion of the nuclease domain of Exo1 reduces accumulation of DNA damage and DNA damage signal induction in telomere-dysfunctional mice. Exo1 deletion improved organ maintenance and lifespan of telomere-dysfunctional mice but did not increase chromosomal instability or cancer formation. Deletion of Exo1 also ameliorated the induction of DNA damage checkpoints in response to gamma-irradiation and conferred cellular resistance to 6-thioguanine-induced DNA damage. Exo1 deletion impaired upstream induction of DNA damage responses by reducing ssDNA formation and the recruitment of Replication Protein A (RPA) and ATR at DNA breaks. Together, these studies provide evidence that EXO1 contributes to DNA damage signal induction in mammalian cells, and deletion of Exo1 can prolong survival in the context of telomere dysfunction.     

Expanded bed adsorption in the purification of monoclonal antibodies: a comparison of process alternatives

Expanded bed adsorption (EBA) was examined as the initial capture/purification step in the purification of monoclonal antibodies from Chinese hamster ovary (CHO) cultures. Two process alternatives each using EBA were compared to a conventional Protein A process without EBA. One alternative used Protein A affinity EBA followed by packed-bed cation and anion-exchange steps. The other alternative used cation-exchange EBA as the capture step followed by packed-bed Protein A and anion-exchange steps. The process using Protein A EBA produced comparable purity (host cell protein, DNA, Protein A, antibody aggregate) to the conventional process. However, the Protein A EBA column showed a significant decrease in dynamic capacity with a limited number of cycles. The process using cation EBA achieved comparable levels of host cell proteins (HCP) and DNA but not antibody aggregate or leached Protein A compared to the conventional process.     

Evolution de l'activite auxines-oxydasique et peroxydasique lors de l'induction photoperiodique et de la sexualisation de l'epinard

(Evolution of the auxin-oxidase and peroxidase activity duringthe spinach's photo-periodic induction and sexualisation) Protein extracts, prepared from spinach leaves, are analysedafter a gel chromatography on Sephadex G-100 and SE-SephadexC-50. The photoperiodic induction seems related with a fallof the auxin-oxidase and peroxidase activities we observe achange in the isozyme number and pattern. The female plants have more isozymes, but the enzymatic activityis higher in the male plants. The destruction of the indole-3-acetic acid, in spinach, isnot strictly related to the peroxidases. (Received September 14, 1971; )     

Glycyrrhizin exhibits potential chemopreventive activity on 12-O-tetradecanoyl phorbol-13-acetate-induced cutaneous oxidative stress and tumor promotion in swiss albino mice

Glycyrrhizin and its aglycone, glycyrrhetic acid has been found useful for various therapeutic purposes. Glycyrrhizin has been shown to possess many physiological functions like anti-inflammatory activity, detoxification and inhibition of carcinogenic promoters. 12-O-Tetradecanoyl phorbol-13-acetate (TPA), a well-known phorbal ester is known for its tumor promotion activity. The induction of inflammation in skin mediated by TPA is believed to be governed by cyclooxygenase (COX), lipoxygenase and ornithine decarboxylase (ODC). These markers of inflammatory responses are important for skin tumor promotion. In our present study, we studied the chemopreventive effect of glycyrrhizin on TPA (20 nmol/0.2 mL acetone/animal, topically)-induced oxidative stress and hyperproliferation markers in skin. TPA enhanced lipid peroxidation with reduction in the level of catalase, glutathione, glutathione peroxidase, glutathione reductase and glutathione-s-transferase. TPA treatment also enhanced ODC activity and [³H] thymidine incorporation into cutaneous DNA. Prophylactic treatment of mice with glycyrrhizin (2.0 & 4.0 mg/0.2 mL acetone/animal, topically) resulted in a significant decrease in cutaneous microsomal lipid peroxidation (P < 0.001) and recovery of cutaneous glutathione content (P < 0.001) and its dependent enzymes. A significant inhibition in ODC activity and DNA synthesis (P < 0.001) was also observed. Thus, the results demonstrate that pretreatment with glycyrrhizin is protective against TPA-induced oxidative stress and tumor promotion in Swiss albino mice.     

Functional expression in Escherichia coli of proteins B and C from soluble methane monooxygenase of Methylococcus capsulatus (Bath).

Methylococcus capsulatus (Bath) uses a soluble methane monooxygenase (sMMO) to catalyse the oxidation of methane to methanol. sMMO is comprised of three components; A, B and C. Protein C (the reductase) transfers electrons from NADH to protein A (the hydroxylase) which contains the active site, and protein B regulates this electron flow. The five genes encoding the sMMO proteins and their subunits are clustered and have been cloned in Escherichia coli. A DNA fragment containing mmoB, the gene encoding protein B, was subcloned into pT7-5, a plasmid of the T7 RNA polymerase promoter expression system. Upon induction, E. coli expressed protein B which was fully functional after purification. The gene encoding protein C, mmoC, was amplified with unique restriction sites at each end using the polymerase chain reaction and then subcloned into pT7-7 (a plasmid similar to pT7-5 but containing its own ribosome-binding site and ATG start codon). Protein C expressed in E. coli was also found to be functional. This is the first report of the functional expression of methanotroph methane monooxygenase genes in a heterologous host and represents a significant step forward in our analysis of the assembly and catalysis of sMMO.