Tyrosine phosphorylation controls cortactin binding to two enterohaemorrhagic Escherichia coli effectors: Tir and EspFu/TccP

Enterohaemorrhagic Escherichia coli (EHEC) is an important food-borne pathogen that, upon infection, causes destruction of the microvilli brush border of intestinal cells. EHEC is able to recruit several host cell proteins and induce actin accumulation beneath its adherence site, forming a pedestal-like structure upon which the bacterium is firmly attached. Injection of bacterial effectors into the host cells is required to trigger the recruitment and activation of proteins, such as cortactin, neural Wiskott-Aldrich syndrome protein (N-WASP) and Arp2/3 complex, directly involved in the actin polymerization process. We found that cortactin, an actin-binding protein, has a pivotal role during pedestal formation by EHEC. Cortactin was found to bind directly to two important virulence factors of EHEC, Tir and EspF(u), which are translocated into the host cells during infection. Binding of cortactin to these effectors is dependent upon tyrosine phosphorylation and a balance between tyrosine phosphorylation and dephosphorylation of cortactin is required to regulate pedestal formation by EHEC.     

Riboflavin uptake by the human-derived liver cells Hep G2: Mechanism and regulation

The water-soluble vitamin riboflavin (RF) plays a critical role in many metabolic reactions, and thus, is essential for normal cellular functions and growth. The liver plays a central role in normal RF metabolism and is the site of maximal utilization of the vitamin. The mechanism of liver uptake of RF has been studied in animals, but no information is available describing the mechanism of the vitamin uptake in the human situation and its cellular regulation. In this study, we used the human-derived liver cells Hep G2 as an in vitro model system to address these issues. Uptake of RF by Hep G2 cells was found to be temperature- and energy-dependent but Na⁺-independent in nature. Uptake seemed to involve a carrier-mediated process as indicated by the saturation as a function of substrate concentration (apparent K_m 0.41 ± 0.08 μM), and by the ability of the structural analogs lumiflavin and lumichrome to inhibit the uptake process [inhibition constant (K_i) of 1.84 and 6.32 μM, respectively]. RF uptake was energy dependent, and was inhibited by the -SH group blocker p-chloromercuriphenylsulfonate (p-CMPS) (K_i of 0.10 mM). Specific modulators of intracellular protein kinase A (PKA)-, protein kinase C (PKC)-, and protein tyrosine kinase (PTK)-mediated pathways did not affect RF uptake by Hep G2 cells. On the other hand, specific inhibitors of Ca²⁺/calmodulin-mediated pathway significantly inhibited the uptake process; this effect seemed to be mediated through a decrease in the V_max of the substrate uptake process. Maintaining Hep G2 cells in a RF-deficient growth medium was associated with a significant up-regulation in the substrate uptake; this effect was specific for RF and was mediated mainly by means of an increase in the V_max of the uptake process. These results describe, for the first time, the mechanism and cellular regulation of RF uptake by a human-derived liver cellular preparation, and shows the involvement of a carrier-mediated system in the uptake process. Furthermore, the uptake process seems to be regulated by an intracellular Ca²⁺/calmodulin-mediated pathway and by extracellular substrate levels. J. Cell. Physiol. 176:588–594, 1998. © 1998 Wiley-Liss, Inc.     

Fluctuation in amino acids content in Triticum aestivum L. cultivars as an indicator on the impact of post-emergence herbicides in controlling weeds

This study was carried out in a demonstrated field in El-Sharkia Governorate, Egypt, during the winter of season 2020 to evaluate the leverage of four post-emergence herbicides i.e., tribenuron-methyl, clodinafop- propargyl, pyroxsulam and pinoxaden compared to control on total protein and amino acid contents in three wheat cultivars (Shandwel 1, Giza 171, and Sakha 95). Generally, the use of foliar herbicides led to a significant decrease in essential, non-essential amino acids and protein contents. However, tribenuran-methyl herbicide signifcantly increased the levels of proline, glycine, arginine, and histidine, but cystine and threonine not affected as compared to control. On the other hand, foliar herbicide application was significantly increased physiological , biochemical parameters and yield of Shandweel cultivar as compared to the other varieties. The physiological and biochemical models of dual-herbicide-tolerant wheat cultivars add to our understanding of the crop. In recent agricultural systems, herbicide tolerant plants are important for long-term weed management. Therefore, the study recommended the safely usage of Tribenuran-methyl as foliar herbicide in weed managment.     

Dilated cardiomyopathy in Erb-b4-deficient ventricular muscle

The neuregulin receptor tyrosine kinase Erb-b4, initially linked to early cardiac development, is shown here to play a critical role in adult cardiac function. In wild-type mice, Erb-b4 protein localized to Z lines and to intercalated disks, suggesting a role in subcellular and intercellular communications of cardiomyocytes. Conditional inactivation of erb-b4 in ventricular muscle cells led to a severe dilated cardiomyopathy, characterized by thinned ventricular walls with eccentric hypertrophy, reduced contractility, and delayed conduction. This cardiac dysfunction may account for premature death in adult erb-b4-knockout mice. This study establishes a critical role for Erb-b4 in the maintenance of normal postnatal cardiac structure and function.     

Comparative study on the susceptibility of cutworms (Lepidoptera: Noctuidae) to Agrotis segetum nucleopolyhedrovirus and Agrotis ipsilon nucleopolyhedrovirus

The common cutworm (Agrotis segetum) and the black cutworm (Agrotis ipsilon) are serious soil pests of many vegetable and field crops all over the world. We have demonstrated the cross-infectivity of two baculoviruses, A. segetum nucleopolyhedrovirus (AgseNPV) and A. ipsilon nucleopolyhedrovirus (AgipNPV) for these two insect pests. The susceptibility of A. segetum to AgipNPV was confirmed by DNA restriction endonuclease analyses of DNA isolated from virus harvested from infected A. segetum larvae. For an initial comparison of both viruses, partial polyhedrin sequences were amplified by PCR, cloned, and sequenced. Both viruses shared a very similar polyhedrin gene sequence resulting in only three amino acid substitutions. Phylogenetic analyses clearly demonstrated that both viruses belong to NPV group II and are most closely related to a clade consisting of Spodoptera exigua NPV, Spodoptera frugiperda NPV, and Spodoptera littoralis NPV. Since AgipNPV shows high virulence for both cutworm species, it appears to be a suitable candidate as a single biological control agent of A. segetum and A. ipsilon.     

HCV/HBV复合抗原基因的克隆、表达及免疫应答研究

为探讨ＨＣＶ／ＨＢＶ 复合疫苗的可行性,将合成的丙型肝炎病毒（ＨＣＶ）复合多表位抗原基因ＰＣＸ与ＨＢｓＡｇ 基因连接成ＰＣＸＳ基因,与β－半乳糖苷酶（ＧＺ）基因融合后在大肠杆菌及减毒鼠伤寒沙门氏菌中获得表达．目的蛋白ＧＺ－ＰＣＸＳ可被抗－ＨＢｓ 及抗－ＨＣＶ 抗体所特异识别．ＧＺ－ＰＣＸＳ抗原皮下注射免疫ＩＣＲ小鼠后,诱发了较高水平的抗－ＧＺ－ＰＣＸＳＩｇＧ反应．构建的重组减毒鼠伤寒沙门氏菌ＳＬ３２６１（ｐＷＲ／ＰＣＸＳ）口服免疫小鼠后,诱发了高水平的ＣＤ８＋ Ｔ细胞增殖反应及抗ＧＺ－ＰＣＸＳＩｇＧ反应．所有免疫小鼠均未见明显的毒副作用．该研究揭示,ＨＣＶ／ＨＢＶ 复合抗原可诱发特异性体液免疫及细胞免疫应答,而活菌苗口服可能是理想的免疫途径,为ＨＣＶ／ＨＢＶ 双价疫苗研究提供了一定的理论及实验依据．     

Breast cancer. Cyr61 is overexpressed, estrogen-inducible, and associated with more advanced disease

To identify genes involved in breast cancer, polymerase chain reaction-selected cDNA subtraction was utilized to construct a breast cancer-subtracted library. Differential screening of the library isolated the growth factor-inducible immediate-early gene Cyr61, a secreted, cysteine-rich, heparin binding protein that promotes endothelial cell adhesion, migration, and neovascularization. Northern analysis revealed that Cyr61 was expressed highly in the invasive breast cancer cell lines MDA-MB-231, T47D, and MDA-MB-157; very low levels were found in the less tumorigenic MCF-7 and BT-20 breast cancer cells and barely detectable amounts were expressed in the normal breast cells, MCF-12A. Univariate analysis showed a significant or borderline significant association between Cyr61 expression and stage, tumor size, lymph node positivity, age, and estrogen receptor levels. Interestingly, expression of Cyr61 mRNA increased 8- to 12-fold in MCF-12A and 3- to 5-fold in MCF-7 cells after 24- and 48-h exposure to estrogen, respectively. Induction of Cyr61 mRNA was blocked by tamoxifen and ICI182,780, inhibitors of the estrogen receptor. Stable expression of Cyr61 cDNA under the regulation of a constitutive promoter in MCF-7 cells enhanced anchorage-independent cell growth in soft agar and significantly increased tumorigenicity and vascularization of these tumors in nude mice. Moreover, overexpression of Cyr61 in MCF-12A normal breast cells induced their tumor formation and vascularization in nude mice. In summary, these results suggest that Cyr61 may play a role in the progression of breast cancer and may be involved in estrogen-mediated tumor development.     

mtDNA analysis of Nile River Valley populations: A genetic corridor or a barrier to migration?

To assess the extent to which the Nile River Valley has been a corridor for human migrations between Egypt and sub-Saharan Africa, we analyzed mtDNA variation in 224 individuals from various locations along the river. Sequences of the first hypervariable segment (HV1) of the mtDNA control region and a polymorphic HpaI site at position 3592 allowed us to designate each mtDNA as being of "northern" or "southern" affiliation. Proportions of northern and southern mtDNA differed significantly between Egypt, Nubia, and the southern Sudan. At slowly evolving sites within HV1, northern-mtDNA diversity was highest in Egypt and lowest in the southern Sudan, and southern-mtDNA diversity was highest in the southern Sudan and lowest in Egypt, indicating that migrations had occurred bidirectionally along the Nile River Valley. Egypt and Nubia have low and similar amounts of divergence for both mtDNA types, which is consistent with historical evidence for long-term interactions between Egypt and Nubia. Spatial autocorrelation analysis demonstrates a smooth gradient of decreasing genetic similarity of mtDNA types as geographic distance between sampling localities increases, strongly suggesting gene flow along the Nile, with no evident barriers. We conclude that these migrations probably occurred within the past few hundred to few thousand years and that the migration from north to south was either earlier or lesser in the extent of gene flow than the migration from south to north.