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Acta Microbiologica et Immunologica Hungarica
Authors: Ghasem Bagherpour, Abbas Fooladi, Jalil Mehrabadi, Mohammad Nourani, and Behzad Einollahi

Uropathogenic Escherichia coli (UPEC) bacteria are the principal cause of urinary tract infections (UTI). Because these bacteria propagate intracellularly, the cellular immune response is an important factor in UTIs. Therefore, we designed a genetic construct to induce a cellular immune response. In order to develop a genetic construct that induces strong cellular immunity against this pathogen, we used the fimH synthetic gene according to mammalian codon usage, and the gene expression was compared with wild type codon usage. Initially, we designed two constructs, pVAX/fimH mam and pVAX/fimH wt, which contain mammalian and wild type codon usage, respectively. The Cos-7 cell line was transfected separately with a complex of pVAX/fimH mam-ExGene 500 poly cationic polymer and pVAX/fimH wt-ExGene 500 poly cationic polymer. Expression of the fimH gene in both constructs in COS7 cells was confirmed by RT-PCR, SDS-PAGE, and Western blotting. Both of the pVAX/fimH cassettes expressed inserted fimH genes (mam and wt) in Cos-7 cells. Our results suggest that codon optimization successfully expressed the fimH gene because the fimH gene with mammalian codon usage is compatible with the eukaryotic expression system. Therefore, mammalian codon usage could be appropriate in a pVAX/fimH construct as a DNA vaccine.

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Acta Physiologica Hungarica
Authors: A Todorović, A Todorović, A Todorović, S Pejić, S Pejić, S Pejić, J Kasapović, J Kasapović, J Kasapović, V Stojiljković, V Stojiljković, V Stojiljković, SB Pajović, SB Pajović, SB Pajović, DT Kanazir, DT Kanazir, and DT Kanazir

In order to examine if differences in activity and inducibility of antioxidative enzymes in rat cerebral cortex and hippocampus are underlying their different sensitivity to radiation, we exposed four-day-old female Wistar rats to cranial radiation of 3 Gy of g-rays. After isolation of hippocampus and cortex 1 h or 24 h following exposure, activities of copper-zinc superoxide dismutase (CuZnSOD), manganese superoxide dismutase (MnSOD) and catalase (CAT) were measured and compared to unirradiated controls. MnSOD protein levels were determined by SDS-PAGE electrophoresis and Western blot analysis. Our results showed that CuZnSOD activity in hippocampus and cortex was significantly decreased 1 h and 24 h after irradiation with 3 Gy of g-rays. MnSOD activity in both brain regions was also decreased 1 h after irradiation. 24 h following exposure, manganese SOD activity in hippocampus almost achieved control values, while in cortex it significantly exceeded the activity of the relevant controls. CAT activity in hippocampus and cortex remained stable 1 h, as well as 24 h after irradiation with 3 Gy of g-rays. MnSOD protein level in hippocampus and cortex decreased 1 h after irradiation with 3 Gy of g-rays. 24 h after exposure, MnSOD protein level in cortex was similar to control values, while in hippocampus it was still significantly decreased. We have concluded that regional differences in MnSOD radioinducibility are regulated at the level of protein synthesis, and that they represent one of the main reasons for region-specific radiosensitivity of the brain.

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Gluten proteins composed of gliadins and glutenins are important contributors to the wheat quality properties. Twenty-eight winter wheat cultivars differing in bread processing quality were collected at the experimental fields of the Agricultural Institute Osijek, Croatia, in growing season 2006/2007.The HMW-GS composition and gliadin contents were determined by SDS-PAGE and RP-HPLC, respectively, with the aim to determine their relationship with wheat quality properties. Based on gliadins and HMW-GS data for 28 wheat cultivars PLS models were developed for the prediction of 15 baking quality parameters.NIPALS algorithm was applied for the evaluation of the latent variables and regression coefficient parameters. The obtained 4-th order models have average coefficients of determination R2=0.80.Determined variable importance in projections (VIP) coefficients revealed that HMW-GS data have the dominant influence on the baking quality parameters. For extensographic and farinographic properties the Glu-D1 locus has the main VIP coefficient while Glu-B1 locus is the most important for the indirect quality parameters. The derived PLS models and VIP coefficients could be used in molecular based wheat selection and breeding program.

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Sodium chloride tolerant organogenic callus lines of Nicotiana tabacum were developed in vitro on Murashige and Skoog [16] medium supplemented with BA, IAA and different concentration of NaCl. The maximum shoot bud regeneration was achieved from both tolerant and non-tolerant calluses on MS medium supplemented with 1.0 mg/l BA, 0.1 mg/l IAA with or without NaCl within 4 weeks of culture. Standard growth parameters such as fresh weight and dry weight of organogenic callus, growth tolerant index and enzyme activity (peroxidase and catalase) were used as indicators of salt tolerance. The growth tolerance index in the 4-week after the beginning of treatments yielded significant differences among the non-tolerant and tolerant organogenic callus lines. The regenerated shoots were rooted on half-strength MS basal salts supplemented with 2% sucrose but devoid of growth regulator. The regenerated plants from tolerant callus lines were capable of growing in vitro in presence of 175 mM NaCl. SDS-PAGE profile showed that the progenies derived from tolerant sources were tolerant to salt. This investigation may help in the selection and characterization of salt tolerance in plant improvement programme.

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Summary  

In the present work, we investigated the immunological behavior of bothropstoxin-1, a K49 phospholipase from Bothrops jararacussu, and of ovalbumin before and after irradiation with 60Co g-rays. Isogenic mice were immunized with either native or irradiated proteins. The circulating antibodies were isotyped and titrated by ELISA. Results indicate that irradiated proteins were immunogenic and the antibodies elicited by them were able to recognize the native proteins in ELISA. Data also indicate that the irradiated protein induced higher titers of IgG2a and IgG2b, suggesting that Th1 cells were predominantly involved in the immune response. Structural modifications of the proteins were investigated by SDS-PAGE, mass spectrometry and size exclusion chromatography. According to our data, irradiation promoted structural modifications on both proteins, characterized by higher molecular weight forms (aggregates and oligomers). When analyzed by mass spectrometry, the irradiated bothropstoxin appeared in several oxidized forms. These results indicate that irradiation of toxic proteins can promote significant modifications in their structures, but still retain many of the original antigenic and immunological properties of native form.

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Pasteurella multocida B:2 is responsible for haemorrhagic septicaemia in cattle and buffaloes, causing severe economic losses in the developing countries. In the present study, the ahpA gene of P. multocida B:2 (P52) was cloned, sequenced and compared with the previously reported ahpA gene sequence in P. multocida A:1, which is responsible for its haemolytic phenotype. E. coli DH5α cells were further transformed with recombinant plasmid carrying the ahpA gene from P. multocida B:2 (P52) but SDS-PAGE analysis failed to show the expression of haemolysin protein. Slight haemolysis was albeit observed in horse blood agar plates streaked with recombinant E. coli carrying the ahpA gene. Our study indicates that there is 99.6% similarity and 0.4% divergence between ahpA gene of P. multocida B:2 (P52) and P. multocida A:1, while membrane topology analysis has predicted that ahpA is an inner membrane protein with two strong hydrophobic regions at the N and C terminals. The presence of significant homology in ahpA sequence in A:1 and B:2 perhaps suggests a common mechanism of pathogenesis in different species of animals.

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Aim of this study is to analyze the effect of chronic administration of beta agonist isoproterenol hydrochloride (60 mg kg −1 day −1 ; 30 days) on soleus (a slow type) and extensor digitorum longus (EDL, a fast type) muscles in young mice. Isoproterenol resulted in significant increase in muscle weight to whole body weight ratio with no increase in hypertrophy index in soleus muscle. A significant increase in noncontractile protein collagen is also observed in both muscles but more prominent in soleus muscle. Collagen proliferation is also analyzed on sodium dodecyle sulphate polyacrylamide gel electrophoresis (SDS-PAGE) of pepsin soluble and Cyanogen Bromide (CNBr) treated pepsin insoluble collagen. Isoproterenol remolded the myofibrillar proteins in both muscles but significant increase in myofibrillar ATPase activity occurred only in soleus muscle. It is concluded that growth stimulatory effect of isoproterenol hydrochloride is more prominent in soleus than EDL muscle. Isoproterenol augmented the proliferation of non-contractile protein collagen in soleus and EDL muscles. The transformation in myofibrillar proteins caused by isoproterenol might lead to an enhancement of contractile performance.

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The objective of this study is the analysis of polymorphism in seed endosperm proteins (gliadins and glutenins) of Turkish cultivated einkorn wheat [Triticum monococcum ssp. monococcum] landraces. The genetic diversity of high-molecular-weight (HMW) glutenin subunits and the gliadin proteins in 10 landrace populations of cultivated einkorn wheat, originating from Turkey, was investigated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and ammonium lactic acid polyacrylamide gel electrophoresis (A-PAGE), respectively. For glutenins, the mean number of alleles, the mean number of effective alleles, the mean value of genetic diversity and the mean value of average genetic diversity were detected as 3.50, 2.98, 0.65 and 0.28, respectively. The genetic differentiation was 0.57, while gene flow was 0.19 between populations. For gliadins, the mean number of alleles, the mean number of effective alleles, the mean value of total genetic diversity and the genetic diversity within population were detected as 2.00, 1.21, 0.17 and 0.15, respectively. The genetic differentiation was 0.08, whereas gene flow was 6.15 between populations. STRUCTURE is a software package program for population genetic analysis, was used to infer population structures of landraces populations. The optimum value for K was obtained as 10. Considering the high number of proteins and genetic variation, and increased interest in organic products, the farming of einkorn wheat should be supported and conservation of germplasm in landraces should be maintained as important genetic resources. The landraces germplasm should be conserved for future crop improvement processes.

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A novel HMW-GS of Dx5** with slightly faster migration rate than that of Dx5, was found in a Tibet bread wheat landrace using SDS-PAGE. Moreover, Dx5** is the subunit with the fastest migration rate in Glu-Dx locus. The gene for this subunit was isolated and its sequence was obtained in the present study. This gene was very similar to Dx5 both in nucleotide and deduced amino acid sequence. At the nucleotide sequence level, Dx5** different from Dx5 by the deletion of a 27 bp fragment and two nucleotide replacements at position 353(G/C) and 692(C/G), respectively. At the amino acid sequence level, Dx5** different from Dx5 by the deletion of a hexaploid (LGQGQQ) and a tripeptide (GQQ) repetitive motif and two amino acid replacements at position 118(C/S) and 231(A/G), respectively. These results suggested that the Dx5** was a derivation of Dx5 and was formed by replication slippage. Moreover, the specific cysteine (C) located at the beginning of the repetitive domain of Dx5, which proved to be critical for the end-use quality of wheat flours, was replaced by serine (S) in Dx5**.

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Optimization of extraction ratio (ER) of tree peony seed protein (TPSP) was investigated using response surface methodology (RSM). The second-degree equation for ER of TPSP had high coeffi cient (0.9625) of determination. The probability (P) value of regression model signifi cance was less than 0.001 by analysis of central composite rotatable design. Relationships of ER to pH, liquid/solid ratio, squares of all factors, and cross-product factors (x2x3, x2x4, x3x4) were signifi cant (P<0.05). Whereas, extraction time, temperature, and cross-product terms (x1x2, x1x3, x1x4) were not signifi cant factors (P>0.05). Optimum extraction conditions were 3.42 h, pH 9.50, 50.80 ºC, and 9.54 ml g–1 of liquid/solid ratio with the maximum ER (43.60%) . SDS-PAGE indicated TPSP had mainly four proteins (180, 100, 60, and 35 kDa) with four subunits of 60, 48, 38, and 23 kDa. TPSP had a good amino acid composition with abundant essential amino acids (39.76%) determined by amino acid analysis.

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