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To construct a prokaryotic promoter report system with wide applicability, a series of pFGH reporter vectors based on lacZ gene and pUC replicon were constructed from plasmid pFLX107 through the replacement of multiple cloning sites and sequence modifications. The plasmid with the lowest background activity was selected as the final report system with the lacZ gene deletion strain MC4100 as the host bacterium, following by testing with inducible promoter araBAD and the constitutive promoter rpsM. The background activity of pFGH06 was significantly lower than that of other plasmids of the same series, and even lower than that of reference plasmid pRCL at 28 °C (P<0.01). Further evaluation tests show that the plasmid pFGH06 could be used to clone and determine the activity of inducible promoter or constitutive promoter, and the complete recognition of the target promoter could be achieved through blue-white selection in the simulation test of promoter screening. Compared with the reported prokaryotic promoter report systems, pFGH06 has the advantages of smaller size, more multiple clone sites, adjustable background activity, high efficiency of promoter screening and recognition, thus with a wide application prospect.
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Cloning, Molecular , Escherichia coli/genetics , Genes, Reporter/genetics , Genetic Vectors/genetics , Lac Operon/genetics , Plasmids/genetics , beta-Galactosidase/geneticsABSTRACT
OBJECTIVE: To observe the effects of lung injury caused by sodium arsenite in male rats,and to explore its cell senescence mechanism. METHODS: Specific pathogen free healthy adult male Wistar rats were randomly divided into 4groups with 8 rats in each group: a control group( given ultrapure water) and low-,medium- and high-sodium arsenite dose groups( 10,100 and 1 000 μg / L sodium arsenite in drinking water,respectively). The rats were euthanized after 4weeks of treatment. The pathologic changes in the lung were examined and the levels of interleukin( IL)-1β,IL-6,IL-10 and β-galactosidase( β-Gal) in broncho-alveolar lavage fluid( BALF) were determined by enzyme-linked immunosorbent assay. RESULTS: The lung tissue of the 3 treatment groups showed early inflammatory pathological changes. With the increase of dose,lung histopathology gradually showed the alveolar interval widened,infiltrating with protein-rich fluid and a large number of inflammatory cells. The levels of IL-1β,IL-6 and β-Gal in BALF of the medium- and high-dose groups were higher than those of the control group( P < 0. 05). The levels of IL-10 in BALF of the 3 treatment groups were lower than that of the control group( P < 0. 05). The levels of β-Gal and IL-1β in BALF of rats as well as with IL-6 was positively correlated [the correlation coefficient( r) were 0. 691 and 0. 410 respectively,P < 0. 05]. The levels of β-Gal and IL-10 were not correlated( r =- 0. 117,P > 0. 05). CONCLUSION: Sodium arsenite in drinking can result in inflammatory injury in lung in male rats. The mechanism may be related to the cell senescense that activates the inflammatory cascade.
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Aims: To evaluate utility of replication system using chromogenic disc for detection of uropathogens and to compare the result with conventional method for the same. Design: A total of 625 urine samples were processed from suspected cases of urinary tract infection, admitted in a rural medical college of Maharashtra. Methodology: The culture isolates of uropathogens were identified by both conventional method and by chromogenic disc using replica system. Results: Out of 625 urine specimens, 419 (67.04%) were Culture positive. There was growth of Escherichia coli 197(99.49%), Enterococcus faecalis 88 (21%), Klebsiella pneumoniae 63 (15.03%) and Pseudomonas aeruginosa 10.73%. There was mixed growth of organisms 15(3.57%) in urine specimens. All uropathogens isolates were identified correctly by conventional as well as chromogenic disc using replica system, except one. One of the Escherichia coli isolate was identified by conventional methods but with replica system it showed colourless colonies instead of purple colonies. Conclusion: Replica system is a rapid, cost effective and easy method for detection of uropathogens with satisfactory result. It can be adopted in clinical microbiology laboratory for presumptive diagnosis of uropathogens.
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Background Researchers are paying increasing attention to the effect of cellular senescence in vascular dysfunctional diseases,and it is hypothesized that cellular senescence may also be involved in the development of diabetes related vascular complications.The outstanding feature of cellular senescence is the upregulation of beta-galactosidase.Objective This study was to investigate the effects of high glucose on cell senescent in vitro and in vivo on bovine retinal endothelial cells (BRVECs) and mouse retina.Methods BRVECs were cultured and passaged,and the seventh generation of cells were employed in this study.The cells were divided into the control group and the high glucose culture group and cultivated using M199 medium containing 5.5 mmol/L or 25.0 mmol/L glucose,respectively.5-bromine-chlorine-4-3-indole-beta D galactose glucoside (X-Gal) staining was used to examine the expression of beta-galactosidase in the cells.Diabetic models were established in the SPF male C57BL/6J mice aged 8-10 weeks by intraperitoneal injection of streptozotocin (STZ),and the age-and gendermatched normal mice served as controls.The mouse retinas were collected and starched in the 48-well plates 3 months later.X-Gal staining was employed to calculate the positive cells.Results BRVECs grew well 24 hours after culture but showed irregular arrangement.Forty-eight hours later,the cells reached confluence with a tight connect.The ratios of positive BRVECs and total cells were (51.4±5.4) ％ and (36.6-±3.8) ％ in the high glucose culture group and the control group,with a significant difference between the two groups (t =-3.204,P =0.033).The number of positive cells for X-Gal in mouse retinas was (94.0± 15.1) /field in the diabetic group,which was higher than that in the control group ([60.0 ± 5.7]/field) (t =-2.974,P =0.041).Conclusions High glucose environment accelerates senescence of retinal cells,and high glucose induces premature cell senescence,which likely plays an important role in the pathogenesis of diabetic retinopathy.
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Aim: Production of lactulose and other oligosaccharides by Lactobacillus acidophilus NRRL 4495 β-galactosidase and their biological activity. Methodology and Results: The transgalactosylation activity of Lactobacillus acidophilus NRRL 4495 β-galactosidase was investigated under different conditions for synthesis of lactulose and oligosaccharides. The synthesis was optimized with respect to pH; time; enzyme concentration and substrates ratio (lactose: fructose). Maximum production for lactulose was found to be 25 g/L at pH 6.6 with 40: 20% (w/v) lactose to fructose, respectively and enzyme concentration 4 IU/mL after 7 h. With respect to the other oligosaccharides the maximum yield (19 .68 g/L) was obtained under the same conditions but with enzyme concentration 2 IU/mL and after 10 h. As a new pharmaceutical application the produced lactulose and oligosaccharide and their sulfated derivative were found to have fibrinolytic activity, but they failed to act as anticoagulant. Conclusion significance and impact of study: the research leads to increase the production of lactulose and other oligosaccharides with a significant yield and discovered a new pharmaceutical application for all the products.
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β-galactosidases enzyme have been used in the dairy industry for the improvement of lactose intolerance. The aim of the present study was to isolate β-galactosidase enzyme produced by isolated lactobacillus from milk and cheese. Isolated lactobacilli were cultured on MRS agar. Lactobacilli were identified by Gram staining and standard bacteriological and biochemical methods. Their ability to hydrolyze 5-bromo-4-chloro-3-indolyl-β-Dgalactopyranoside (X-Gal) and O-nitrophenyl- β-Dgalactopyranoside (ONPG) was determined. β-galactosidase enzyme activity was also detected by Sodium Dodecyl Sulphate Gel electrophoresis (SDS-PAGE) method. The colonies that produced blue green color on X-Gal plates were lactobacillus with β-galactosidase enzyme which had ONPG positive results. By adding Lactobacillus producing β-galactosidase enzyme as probiotic to dairy products, could help lactose intolerant infants.
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Objective To study the effect of multiple IPL treatment on cell senescence markers of skin fibroblasts using UVA as a control and to make clear whether the multiple IPL treatment may result in cell senescence.Methods Cells were divided into three groups: one group without irradiation as a control,one group receiving IPL treatment with 15 J per cm2,and the last group receiving UVA irradiation with 9 J per cm2.IPL and UVA irradiation were performed once a day during five days.On the sixth day,the cells were collected.Senescence-associated β-galactosidase (SA-β-Gal) staining,cell cycle,reactive oxygen species (ROS) and telomere length were determined.Results Our results showed that five consecutive days of IPL irradiation had no effect on the activity of SA-β-Gal and telomere length and decreased the G1 ％ of cell cycle and the level of ROS in comparison with the control group (P＜0.05).On the contrary,five consecutive days of UVA irradiation increased the activity of SA β Gal and the level of ROS,shortened the length of telomere and no obvious change in the G1 ％ of cell cycle in comparison with the control group.Conclusions Multiple UVA irradiations induce cell senescence.On the contrary,multiple IPL treatments could not induce cell senescence.
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Objective To investigate the changes in morphology and senescence-associated markers of the marrow-derived cardiac stem cells (MCSCs) from rats at different ages and to explore the impacts of age on proliferation, survival and differentiation of MCSCs. Methods With single-cell cloning culture, MCSCs were selected from the bone marrow of young, adult and aged male SD rats respectively. Ultrastructural changes of the cells were viewed under a transmission electron microscope.The senescence-associated changes were examined with SA-β-galactosidase staining and reactive oxygen species(ROS) staining. Distribution of cell cycle of MCSCs from different age groups was evaluated with flow cytometric analysis. Rates of the survived and apoptotic cells were determined by Annexin V/PI double-labeled flow cytometric analysis and Hochest33342 staining. Differentiation of the MCSCs toward cardiomyocytes was induced with BMP-2. Expression of cardiac transcription factors and cardiac specific genes of the cells after induction were examined with RT-PCR. CTnT expression of the cells also be examined with immunocytochemistry. Results The nucleus/plasma ratio of the cells from aged rats decrease and there are some myelin bodies in the cells of aged group. With increasing of age, the MCSCs in S+G2/M phase reduce, while β-galactosidase-positive cells and ROS-positive cells increase. Survival rate of the cells from aged rats is lower than that of the cells from young rats. At four week after induction with BMP-2, expression of Nkx2.5, GATA-4, cTnT mRNA and Cx-43 mRNA of the cells of young group increase significantly. In adult and aged group, expression of the cardiac transcription factors and cardiac specific genes is lower than that of the cells in young group. In immunocytochemical staining, cTnT expression of the cells in young group is stronger after induction with BMP-2. As compared with that of the cells in young group, cTnT expression of the cells in aged group is weak after induction. Conclusion With increasing of age, MCSCs show senescent changes, including their abilities of proliferation, survival and differentiation toward cardiomyocytes decrease.
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Objective To investigate the effect of rosiglitazone (RGTZ) on anti-oxidation in aging rat kidney. Methods Twenty-four-month-old male Sprague-Dawley rats were randomly divided into three groups (n=10): control group (CON), rosiglitazone group (RGTZ) and caloric restriction group (CR). The CON rats were allowed ad libitum access to feed and tap water.The RGTZ rats received intragastric administration of RGTZ (4 mg·kg-1·d-1),and the CR rats were provided with a vitamin and mineral fortified version of the same diet at a level of 40% less food (by weight) than the CON rats. After 12 weeks all the animals were sacrificed by decapitation, and both the body weight and the percentage of kidney and heart in each group were measured.Western blot was performed to analyze the expression of PPARγ protein. The content of MDA and the activity of SOD and GSH-PX in kidney tissue were detected. Besides, frozen sections of kidney tissue were stained for senescence-associated-13 galactosidase (SA-β-Gal). Results The body weight of CR rats decreased obviously, in contrast, which did not change in CON and RGTZ group. Percentage of kidney and heart to body weight was normal in CR or RGTZ group after intervention. Western blot result showed that PPARγ protein expression in rat kidney was significantly higher in RGTZ and CR group as compared to CON group (P<0.05). Compared with RGTZ and CR rats, obviously lower activities of SOD and GSH-Px were noted in CON rats, however, the content of MDA was higher in CON rats. Additionally, the positive staining area of [3-Gal in CR and RGTZ group was significantly smaller than that in CON rats (P<0.05, P<0.01 ). Conclusion RGTZ can defer the kidney aging in senescence SD rat, and the mechanism may be related to amelioration of oxidative damage and enhancement of antioxidation.
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A Concanavalin A-β-galactosidase conjugate was prepared using glutaraldehyde as the crosslmking reagent. The conjugate bound to Sephadex G-50 beads was more thermostable and hydrolyzed lactose faster than the free enzyme. The immobilized enzyme may prove useful in the preparation of low lactose milk which is required by persons suffering from lactose intolerance.
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A 355 base pair DNA sequence coding for human preproinsulin has been assembled by joining 55 synthetic deoxyoligonucleotide fragments prepared by the modified phosphotriester methodology. Proinsulin was expressed under lac promoter control and truncated β -galactosidase 590 amino acid long sequence. The fused β-galactosidase proinsulin protein was produced in amount to 30 % of the total Escherichia coli proteins. It was also expressed in Μ13 bacteriophage and yeast system.
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b-D-Galactosidase (EC 3.2.1.23) from Lactobacillus bulgaricus (1373) was immobilized in a Polyacrylamide gel lattice in the presence of dithiothreitol, glutathione, cysteine, bovine serum albumin, casein, lactose and glucono-δ-lactone. Cysteine, bovine serum albumin, and lactose were found very effective in preserving the activity. With cysteine, bovine serum albumin and lactose, the activity yields were 61, 60 and 66% respectively, as compared to 31% without protective agents. The yield improved upto 85% when all the three protective agents, cysteine, bovine serum albumin and lactose were added during immobilization. The addition of protective agents did not have any effect on optimum pH, optimum temperature, kinetic constants and pH stability when compared with β-galactosidase immobilized without the use of protective agents; however the heat and storage stabilities were found to increase.
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β-D-galactosidase (EC 3.2.1.23) from Lactobacillus bulgaricus (1373) was immobilized by entrapment in a Polyacrylamide gel lattice. The enzymatic properties of the immobilized β-galactosidase were compared with those of the native enzyme. The temperature and pH optima were not affected by the immobilization. After entrapment of the enzyme no significant change was observed in its thermostability. The pH stability of the immobilized enzyme was higher than that of the native enzyme on the acidic side. The Km values for the immobilized and native β-galactosidase with both lactose and o-nitrophenyl-β-D-galactoside as substrates were comparable. The immobilized enzyme could be repeatedly used 12 times without any loss of activity. No loss in the activity of the immobilized β-galactosidase was found after its storage for 30 days at 4°C and for 20 days at 25°C.