Amount Of Β-galactosidase Research Articles

Oxidative stress induces the decline of brain EPO expression in aging rats

Brain Erythropoietin (EPO), an important neurotrophic factor and neuroprotective factor, was found to be associated with aging. Studies found EPO expression was significantly decreased in the hippocampus of aging rat compared with that of the youth. But mechanisms of the decline of the brain EPO during aging remain unclear. The present study utilized a d-galactose (d-gal)-induced aging model in which the inducement of aging was mainly oxidative injury, to explore underlying mechanisms for the decline of brain EPO in aging rats. d-gal-induced aging rats (2months) were simulated by subcutaneously injecting with d-gal at doses of 50mg·kg−1, 150mg·kg−1 and 250mg·kg−1 daily for 8weeks while the control group received vehicle only. These groups were all compared with the aging rats (24months) which had received no other treatment. The cognitive impairment was assessed using Morris water maze (MWM) in the prepared models, and the amount of β-galactosidase, the lipid peroxidation product malondialdehyde (MDA) level and the superoxide dismutase (SOD) activity in the hippocampus was examined by assay kits. The levels of EPO, EPOR, p-JAK2 and hypoxia-inducible factor-2α (HIF-2α) in the hippocampus were detected by western blot. Additionally, the correlation coefficient between EPO/EPOR expression and MDA level was analyzed. The MWM test showed that compared to control group, the escape latency was significantly extended and the times of crossing the platform was decreased at the doses of 150mg·kg−1 and 250mg·kg−1 (p<0.05). Also, the amount of β-galactosidase and the MDA level in the hippocampus were significantly increased but the SOD activity was significantly decreased (p<0.05, 0.01 and 0.01, respectively). Similar to aging rats, the expressions of EPO, EPOR, p-JAK2, and HIF-2αin the brain of d-gal-treated rats were significantly decreased (p<0.05) at 150mg·kg−1 and 250mg·kg−1. Interestingly, negative correlations were found between EPOR (r=−0.699, p<0.01), EPO (r=−0.701, p<0.01) and the MDA level. These results indicated that aging could result in the decline of EPO in the hippocampus and oxidative stress might be the main reason for the decline of brain EPO in aging rats, involved with the decrease of HIF-2α stability.

Effect of Lipofectin on Antigen-presenting Function and Anti-tumor Activity of Dendritic Cells

Dendritic cells (DC) are professional antigen-presenting cells in the immune system and can induce T cell response against virus infections, microbial pathogens, and tumors. Therefore, immunization using DC loaded with tumor-associated antigens (TAAs) is a powerful method of inducing anti-tumor immunity. For induction of effective anti-tumor immunity, antigens should be efficiently introduced into DC and presented on MHC class I molecules at high levels to activate antigen-specific CD8 + T cells. We have been exploring methods for loading exogenous antigens into APC with high efficiency of Ag presentation. In this study, we tested the effect of the cationic liposome (Lipofectin) for transferring and loading exogenous model antigen (OVA protein) into BM-DC. Methods: Bone marrow-derived DC (EM-DC) were incubated with OVA-Lipofectin complexes and then co-cultured with B3Z cells. B3Z activation, which is expressed as the amount of β-galactosidase induced by TCR stimulation, was determined by an enzymatic assay using β-gal assay system. C57BL/6 mice were immunized with OVA-pulsed DC to monitor the in vivo vaccination effect. After vaccination, mice were inoculated with EG7-OVA tumor cells. Results: BM-DC pulsed with OVA-Lipofectin complexes showed more efficient presentation of OVA-peptide on MHC class I molecules than soluble OVA-pulsed DC. OVA-Lipofectin complexes-pulsed DC pretreated with an inhibitor of MHC class I-mediated antigen presentation, brefeldin A, showed reduced ability in presenting OVA peptide on their surface MHC class I molecules. Finally, immunization of OVA-Lipofectin complexes-pulsed DC protected mice against subsequent tumor challenge. Conclusion: Our data provide evidence that antigen-loading into DC using Lipofectin can promote MHC class I- restricted antigen presentation. Therefore, antigen-loading into DC using Lipofectin can be one of several useful tools for achieving efficient induction of antigen-specific immunity in DC-based immunotherapy.

β-Galactosidase Gene of Thermus thermophilus KNOUC112 Isolated from Hot Springs of a Volcanic Area in New Zealand: Identification of the Bacteria, Cloning and Expression of the Gene in Escherichia coli

To isolate the β-galactosidase producing thermophilic bacteria, samples of mud and water were collected from hot springs of avolcanic area near Golden Springs in New Zealand. Among eleven isolated strains, the strain of KNOUC112 produced the highest amounts of β-galactosidase at 40 h incubation time (0.013 unit). This strain was aerobic, asporogenic bacilli, immobile, gram negative, catalase positive, oxidase positive, and pigment producing. Optimum growth was at 70-72°C, pH 7.0-7.2, and it could grow in the presence of 3% NaCl. The main fatty acids of cell components were iso-15:0 (30.26%), and iso-17:0 (31.31%). Based on morphological and biochemical properties and fatty acid composition, the strain could be identified as genus Thermus, and finally as Thermus thermophilus by phylogenetic analysis based on 16S rRNA sequence. So the strain is designated as Thermus thermophilus KNOUC112. A gene from Thermus thermophilus KNOUC112 encoding β-galactosidase was amplified by PCR using redundancy primers prepared based on the structure of β-galactosidase gene of Thermus sp. A4 and Thermus sp. strain T2, cloned and expressed in E. coli JM109 DE3. The gene of Thermus thermophilus KNOUC112 β-galactosidase(KNOUC112β-gal) consisted of a 1,938 bp open reading frame, encoding a protein of 73 kDa that was composed of 645 amino acids. KNOUC112β-gal was expressed as dimer and trimer in E. coli JM109 (DE3) via pET-5b. (Asian-Aust. J. Anim. Sci. 2004. Vol 17, No. 11 : 1591-1598)

Characterization of an oxygen-dependent inducible promoter system, the nar promoter, and Escherichia coli with an inactivated nar operon

The nar promoter of Escherichia coli, which is maximally induced under anaerobic conditions in the presence of nitrate, was characterized to see whether the nar promoter cloned onto pBR322 can be used as an inducible promoter. To increase the expression level, the nar promoter was expressed in E. coli where active nitrate reductase cannot be expressed from the nar operon on the chromosome. A plasmid with the lacZ gene expressing beta-galactosidase instead of the structural genes of the nar operon was used to simplify an assay of induction of the nar promoter. The following effects were investigated to find optimal conditions: methods of inducing the nar promoter, optimal nitrate and molybdate concentrations maximally inducing the nar promoter, the amount of expressed beta-galactosidase, and induction ratio (specific beta-galactosidase activity after maximal induction/specific beta-galactosidase activity before induction). The following results were obtained from the experiments: induction of the nar promoter was optimal when E. coli was grown in the presence of 1% nitrate at the beginning of culture; expression of beta-galactosidase was not affected by molybdate; the induction ratio was maximal, approximately 300, when the overnight culture was grown in the flask for 2.5 h (OD(600) is congruent to 1.3) before being transferred to the fermentor; the amount of beta-galactosidase per cell and per medium volume was maximal when E. coli was grown under aerobic conditions to OD(600) = 1.7; then the nar promoter was induced under microaerobic conditions made by lowering dissolved oxygen level (DO) to 1-2%. After approximately 6 h of induction, OD(600) became 3.2 and specific beta-galactosidase activity became 36,000 Miller units, equivalent to 35% of total cellular proteins, which was confirmed from sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Production and purification of an extracellular β-galactosidase from the Dutch elm disease fungusOphiostoma novo-ulmi

The causal agents of Dutch elm disease, Ophiostoma ulmi (isolate H200) and Ophiostoma novo-ulmi (isolate CKT-11), secreted similar amounts of β-galactosidase in liquid shake cultures when grown on galacturonic acid or sodium pectate (1.45 ± 0.16 and 1.03 ± 0.24 nkat∙mL−1for O. ulmi, respectively, and 1.30 ± 0.08 and 1.28 ± 0.26 nkat∙mL−1for O. novo-ulmi, respectively). Rhamnose and pectin also stimulated secretion but to a lesser extent, whereas on glucose, enzyme activity was barely detectable (≤0.01 nkat∙mL−1). Ophiostoma novo-ulmi was shown by Q-Sepharose chromatography to form two β-galactosidases, named β-galactosidases I and II. In cultures grown on galacturonic acid β-galactosidase I accounted for approximately 75% of the total activity in the culture filtrate. β-Galactosidase I was further purified to apparent electrophoretic homogeneity by means of Sephacryl gel filtration chromatography, chromatofocusing, and Superdex75 gel filtration. The molecular mass of the enzyme was 135 kDa by SDS–PAGE and 123 kDa by gel filtration. Its isoelectric point, determined by chromatofocusing, was 4.9. The optimal pH for enzyme activity was 5.8 and the optimal temperature was 50 °C. The Kmvalues for p-nitrophenyl β-D-galactopyranoside and lactose were 7.52 and 14.23 mM, respectively, and the maximum velocities for these substrates were 1733 and 355 nkat∙mg protein−1, respectively. The Kivalue for D(−)-galactonic acid γ-lactone was 2.29 mM.Key words: Dutch elm disease, β-galactosidase, Ophiostoma ulmi, Ophiostoma novo-ulmi.

POSEX: vectors for osmotically controlled and finely tuned gene expression in Escherichia coli

Expression of the pro U operon of Escherichia coli is directly proportional to the osmolarity of the growth medium. The basal level of proU transcription is very low, but a large increase is triggered by a sudden rise in the external osmolarity. This increased expression is maintained for as long as the osmotic stimulus persists. We have capitalized upon these regulatory features of the proU operon and have constructed a series of expression vectors (pOSEX) permitting osmotically controlled expression of heterologous genes governed by regulatory signals of proU. The pOSEX vectors carry the pro U promoter, an upstream region required for high-level expression, and part of the first structural gene (proV), which acts as a silencer and is necessary to maintain low-level expression in low osmolarity media. An extended multiple cloning site (MCS) positioned at the 3' end of proV' permits the cloning of heterologous genes into the pOSEX plasmids, and efficient transcription terminators derived from the rrnB operon prevent deleterious read- through transcription into the vector portion. The properties of the pOSEX expression vectors were tested by positioning a promoterless lacZ (encoding β-galactosidase) gene from E. coli and the gcdA (encoding carboxytransferase) gene from the Gram+ bacterium Acidaminococcus fermentans under the control of the proU regulatory region. Efficient, osmoregulated and finely tuned expression of both lacZ and gcdA was achieved, and the amount of β-galactosidase and carboxytransferase synthesized were simply controlled by adjusting the osmolarity of the growth medium with various concentrations of NaCl.

Influence of the helper virus on expression of β-galactosidase from a defective HSV-1 vector, pHSVlac

Defective herpes simplex virus type 1 (HSV-1) vectors can deliver genes into both mitotic and postmitotic cells, including neurons and these vectors, therefore, have great potential use. pHSVlac, the prototype HSV-1 vector, expresses the E. coli Lac Z gene from the HSV-1 immediate early 4 5 promoter. pHSVlac can stably express β-galactosidase in a range of mammalian cell lines and in neurons from throughout the nervous system, both in culture and in the adult rat brain. Thus, HSV-1 vectors may be useful for studying HSV-1 latency, neuronal physiology, and performing gene therapy for neurological conditions. A virus stock of pHSVlac consists of identical HSV-1 particles containing either pHSVlac DNA or the HSV-1 helper virus DNA. Thus, a cell can be infected with the pHSVlac virus, the helper virus, or both; consequently, it is important to determine if the helper virus influences the behavior of pHSVlac. The effect of the helper virus on expression of β-galactosidase from pHSVlac was investigated: it was demonstrated first, that pHSVlac can be efficiently packaged into HSV-1 virus particles using each of five HSV-1 temperature sensitive (ts) mutants as helper virus. Second, pHSVlac grown with each of the five HSV-1 ts mutants expressed high levels of β-galactosidase. Third, pHSVlac grown with HSV-1 strain 17 ts K as helper virus expresses the same amount of β-galactosidase in the absence or presence of ts K. Thus, pHSVlac can efficiently express a gene independent of the HSV-1 helper virus.

Initiation, elongation and inactivation of lac messenger RNA in Escherichia coli studied by measurement of its β-galactosidase synthesizing capacity in vivo

The enzyme-forming capacity of lac messenger RNA in Escherichia coli was followed in vivo after induction with isopropyl thiogalactoside by measuring the difference between the amount of β-galactosidase at the moment of de-induction and after full expression of preformed messenger. It was shown that rifampicin stops the process at the same step as de-induction by inducer removal and is without detectable effect on subsequent expression of preformed messenger RNA. lac Messenger RNA initiation seems to occur randomly in time without detectable periodicity. The rate of elongation of messenger RNA was measured by the combined use of rifampicin and of actinomycin D. The synthesizing capacity is due to four classes of messenger RNA: terminated or non-terminated, capable or not capable of initiating new peptide chains. A study of the ratios of these four classes of messenger during steady-state of induced synthesis gave results consistent with a model where inactivation of messenger RNA occurs randomly in time at the initiating end and does not affect protein synthesis by pre-engaged ribosomes. Moreover, the times of transcription and of translation of a given segment of messenger RNA are equal within experimental error. The changes occurring in the synthesizing capacity of messenger RNA were examined under a variety of conditions where translation was inhibited. Initiation and elongation of messenger RNA were not inhibited by 5-methyl-tryptophan, by multiple amino-acid starvation or by puromycin. Thus, there appears to be no obligatory coupling between transcription and translation. However 5-methyltryptophan and multiple amino-acid starvation extended the transcription time while puromyein did not. In contrast, multiple amino-acid starvation and puromycin shortened the half-life of messenger RNA while 5-methyl-tryptophan was without effect on this parameter.

Turnover of protein in Escherichia coli starving for nitrogen

1. 1. Rates of degradation and synthesis. Degradation of pre-labeled protein in starving cells, measured by the exchange into the medium of [ 14C]leucine, proceeds at a rate of 2–3 %/h for 6–8 h at 37°, then abruptly shuts off almost totally. Meanwhile the incorporation of amino acids into peptide bonds, and the amounts of β-galactosidase (EC 3.2.1.23) and alkaline phosphatase (EC 3.1.3.1.) formed, decrease continuously (to less than 0.3 % synthesis of protein/h). Furthermore, at any time, only about 0.05 as much ribosomal protein is made, relative to soluble protein, as is formed in growing cells. 2. 2. Lack of exchange of amino acids in auxotrophs. When an auxotroph is starved for an amino acid as well as for nitrogen, its uptake of other amino acids is severely inhibited. Nevertheless, synthesis of enzyme proteins, as a result of turnover, continues about as well as in cultures starved only for nitrogen. 3. 3. Effect of inhibitors on proteolysis in cells. Degradation of protein is shut off when synthesis of protein is inhibited by chloramphenicol, 2,4-dinitrophenol, 5-methyltryptophan or proflavine at any time during starvation. It continues, however, when amino acid analogues (ethionine, norleucine, p-fluorophenylalanine) are added to the medium, suggesting that a small amount of peptide bond synthesis, but not necessarily of any particular protein, is required for breakdown of protein to continue. 4. 4. It is suggested that the level of amino acids or amino-acyl sRNA may determine both the activity of pre-existing proteases and the permeability to more amino acids in starving cells.