Analysis of immune response: comparison of immunoblots after isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis using cytoplasmic protein extract from Brucella

Analysis of the immune response towards the facultative intracellular bacterium, Brucella melitensis, was studied by immunoblotting after either isoelectric focusing (IEF) or sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). A cytoplasmic extract (CPE) of Brucella melitensis was used as antigen to analyse the response in 17 sera from naturally infected goats. CPE analysed by IEF exhibited 25 proteins within the pH range of 4.35 to 6. Immunoblotting revealed most of the stained bands around pH 4.5-5.4. CPE analysed by SDS-PAGE showed more than 20 silver stained proteins in the molecular range of 16-18 kDa to 70 kDa but immunoblotting revealed only 1 to 6 bands according to the sera tested. Because proteins are preserved in their native state with IEF, in contrast to SDS-PAGE treatment, this technique may be best suited for analysis of the overall response to natural infection.     

Determination of G-protein levels,ADP-ribosylation by cholera and pertussis toxins and the regulation of adenylyl cyclase activity in liver plasma membranes from lean and genetically diabetic (db/db)

Liver plasma membranes prepared from genetically diabetic (db/db) mice expressed levels of G_i α-2, G_i α-3 and G-protein β-subunits that were reduced by some 75, 63 and 73% compared with levels seen in membranes from lean animals. In contrast, there were no significant differences in the expression of the 42 and 45 kDa forms of G_s α-subunits. Pertussis toxin-catalysed ADP-ribosylation of membranes from lean animals identified a single 41 kDa band whose labelling was reduced by some 86% in membranes from diabetic animals. Cholera toxin-catalysed ADP-ribosylation identified two forms of G_s α-subunits whose labelling was about 4-fold greater in membranes from diabetic animals compared with those from lean animals. Maximal stimulations of adenylyl cyclase activity by forskolin (100 μM), GTP (100 μM), p[NH]ppG (100 μM), NaF (10 mM) and glucagon (10 μM) were similar in membranes from lean and diabetic animals, whereas stimulation by isoprenaline (100 μM) was lower by about 22%. Lower concentrations (EC₅₀-60 nM) of p[NH]ppG were needed to activate adenylyl cyclase in membranes from diabetic animals compared to those from lean animals (EC₅₀-158 nM). As well as causing activation, p[NH]ppG was capable of eliciting a pertussis toxin-sensitive inhibitory effect upon forskolin-stimulated adenylyl cyclase activity in membranes from both lean and diabetic animals. However, maximal inhibition of adenylyl cyclase activity in membranes from diabetic animals was reduced to around 60% of that found using membranes from lean animals. Pertussis toxin-treatment in vivo enhanced maximal stimulation of adenylyl cyclase by glucagon, isoprenaline and p[NH]ppG through a process suggested to be mediated by the abolition of functional G_i activity. The lower levels of expression of G-protein β-subunits, in membranes from diabetic compared with lean animals, is suggested to perturb the equilibria between holomeric and dissociated G-protein subunits. We suggest that this may explain both the enhanced sensitivity of adenylyl cyclase to stimulation by p[NH]ppG in membranes from diabetic animals and the altered ability of pertussis and cholera toxins to catalyse the ADP-ribosylation of G-proteins in membranes from these two animals.     

Evidence for the presence of lipopolysaccharide in Treponema phagedenis (biotype Reiterii) but not in Treponema pallidum (Nichols)

Abstract Immunoblotting profiles of whole or protease-K-digested organisms with homologous antisera demonstrated the presence of a characteristic ladder pattern of smooth LPS in Treponema phagedenis . Periodic acid silver staining of SDS-PAGE gels confirmed these findings. However, when heterologous or homologous serum was reacted with Treponema pallidum , no such pattern or cross-reactions were observed. The significance of apparent absence of LPS in T. pallidum is discussed.     

Differentiation of rice varieties by electrophoresis of embryo protein

Summary Variations in the embryo proteins separated by SDS-PAGE have been observed in 43 cultivated varieties of Oryza sativa L. Cluster and discriminant analysis applied to both protein components and morphological characters indicate that knowledge of the differences in embryo proteins can improve our understanding of genetic affinity and make it easier to differentiate between varieties of similar genetic backgrounds.     

Characterization of seed storage protein patterns of Heliotropium digynum

Heliotropium digynum, is a shrub that has ecological importance. The height of the plant differs from one population to another and the difference in length of the inflorescence can be attributed to environmental factors, such as rainfall or type of soil and temperature. To date, no study has shed light on estimation in seed samples of H. digynum in Saudi Arabia. So, the aim is to evaluate and characterize the protein patterns of seed storage proteins of H. digynum to be used as fingerprint of this plant in Saudi Arabia. It is collected from different locations in the central region of Saudi Arabia and total protein extraction from plant was compared in SDS-PAGE. The genetic relationships among all cultivars were analyzed using UPGMA and NJ using Total Lab TL and in the same way using Jaccard Similarity Coefficient dendrogram using STATISTICA (ver.8) software. Results, our data show that amounts of protein are different, although they are of the same type or from the same geographical region. Amounts ranged between 22 and 1.5 mg/g of dry weight. Less amount of protein was obtained from the group of samples collected from Dir’iyah area, and the highest amount of protein was from the group of samples collected from Dyrab area in general.     

Identification of a novel antibacterial protein from hemolymph of freshwater zooplankton Mesocyclops leuckarti

Bacterial infections are the most important problem of health care worldwide. The hemolymph antibacterial proteins of Mesocyclops leuckarti was isolated for the first time and its antibacterial efficacy was evaluated against four different human pathogenic microbes viz., Escherichia coli, Staphylococcus aureus, Klebsiella pneumonia and Shigella flexneri. The antibacterial potential of the antimicrobial proteins of hemolymph samples from plankton cultured in water enriched with Cow Urine Distillate (CUD) was compared with normal ones. The results indicated that the hemolymph proteins were more potential against Gram negative bacteria than Gram positive bacteria. Klebsiella pneumonia was more susceptible to the hemolymph proteins exhibiting a zone of inhibition measuring 27 mm. The supplement of CUD to the culture media further enriched the antibacterial activity of the hemolymph proteins (29 mm). The SDS-PAGE analysis indicated two different types of clear bands representing proteins of 53 kDa and 19 kDa. Overall, this investigation signified that the microcrustaceans have a defence mechanism hemolymph of Mesocyclops leuckarti have a potential agent for novel antibiotics.     

Function of the 23 kDa extrinsic protein of Photosystem II as a manganese binding protein and its role in photoactivation

The function of the extrinsic 23 kDa protein of Photosystem II (PSII) was studied with respect to Mn binding and its ability to supply Mn to PSII during photoactivation, i.e. the light-dependent assembly of the tetramanganese cluster. The extrinsic proteins and the Mn cluster were removed by TRIS treatment from PSII-enriched membrane fragments and purified by anion exchange chromatography. Room temperature EPR spectra of the purified 23 kDa protein demonstrated the presence of Mn. Photoactivation was successful with low Mn concentrations when the 23 kDa protein was present, while in its absence a higher Mn concentration was needed to reach the same level of oxygen evolution activity. In addition, the rate of photoactivation was significantly accelerated in the presence of the 23 kDa protein. It is proposed that the 23 kDa protein plays an important role in providing Mn during the process of PSII assembly and that it acquires Mn during the light-induced turnover of D1 in the PSII damage-repair cycle and delivers Mn to repaired PSII.     

小麦面粉Puroindoline蛋白的提取与纯化

Puroindoline蛋白是小麦面粉中一种非常重要的蛋白质,不仅影响和决定了籽粒的硬度,而且有抗G^＋、G^-菌以及抗真菌的作用。用含4％TritonX-114、100mmol／L pH7．8Tris-HCl缓冲液处理小麦面粉来分离Puroindoline蛋白。经处理后得到的蛋白质混合溶液首先用分子筛葡聚糖G-75纯化,每个收集管内的组分经SDS-PAGE分析,分子量小于31kD的蛋白质组分被回收和集中,回收的蛋白质组分经PEG20000浓缩后,再用离子交换柱羧甲基纤维素（CM-23）进行纯化。其洗脱液分别是双蒸水和NaCl,梯度为0．05～0．7mol／L、8mmol／L pH5．5的MES缓冲液,回收只含15kD的蛋白质的组分,接着用PEG20000浓缩。最后冷冻干燥得到Puroindoline蛋白。     

志贺菌对HeLa细胞侵袭力的研究

通过使用噬斑形成试验、透射电镜及检测志贺菌蛋白表达等方法,研究志贺菌对Hela细胞的侵袭能力。研究发现:37℃培养条件下,胞质内出现成堆的志贺菌;30℃培养条件下,细菌主要分布在细胞外。SDS-PAGE显示,与30℃培养条件相比,在37℃培养下,志贺菌表达蛋白质的种类和数量明显增加。噬斑形成发现,12株福氏志贺菌强毒株,有9株噬斑数>1000个／ml,而2株弱毒株噬斑数则在50个／ml以下。透射电镜证实了志贺菌对细胞的黏附、侵入和释放过程。结果表明:志贺菌对细胞的侵袭能力受温度的影响;不同志贺菌流行株对细胞的侵袭力存在着差异:实验也显示,应用HeLa细胞研究志贺菌侵袭力是一种简便易行且价廉的方法。     

Jasmonic acid induced changes in protein pattern, antioxidative enzyme activities and peroxidase isozymes in peanut seedlings

Protein pattern, ammonia content, glutamine synthetase activity, lipid peroxidation, superoxide dismutase, catalase, peroxidase and peroxidase isoforms were studied in the leaves and roots of 7-d-old peanut (Arachis hypogaea L. cv. JL-24) seedlings treated by 25, 100 and 250 μM jasmonic acid (JA). SDS-PAGE protein profile of leaves and roots after JA application showed a significant increase in 18, 21, 30, 45, 47 and 97.4 kDa proteins and significant decrease in 22 and 36 kDa proteins. Pathogenesis related PR-18 was specific in leaves at 250 μM JA and PR-21 have cross reacted differently with 21 and 30 kDa proteins in leaves and roots treated by all JA concentrations. Further, the immunoblot analysis with glutamine synthetase, GS-45 antibodies revealed a specific cross reaction with 45 and 47 kDa proteins of both control and JA treated leaves, however, higher at 100 and 250 μM JA treated leaves than control ones. Further, the malondialdehyde (MDA) content significantly increased in leaves and roots treated with JA, indicated membrane damage with JA treatments that led to the generation of peroxidation products. The peroxidase isozymic pattern showed two specific isoforms. Besides, the activities of SOD and catalase were significantly elevated in JA treated leaves.