<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated genetic transformation and regeneration of transgenic plants using leaf segments as explants in Valencia sweet orange

In this study, attempts were made to develop a protocol for regeneration of transgenic plants via Agrobacterium tumefaciens-mediated transformation of leaf segments from ‘Valencia’ sweet orange (Citrus sinensis L. Osbeck) using gfp (green fluorescence protein) as a vital marker. Sensitivity of the leaf segments regeneration to kanamycin was evaluated, which showed that 50 mg l⁻¹ was the best among the tested concentrations. In addition, factors affecting the frequency of transient gfp expression were optimized, including leaf age, Agrobacterium concentration, infection time, and co-cultivation period. Adventitious shoots regenerated on medium containing Murashige and Tucker basal medium plus 0.1 mg l⁻¹ α-naphthaleneacetic acid (NAA), 0.5 mg l⁻¹ 6-benzyladenine (BA) and 0.5 mg l⁻¹ kinetin (KT). The leaf segments from 3-month-old in vitro seedlings, Agrobacterium concentration at OD₆₀₀ of 0.6, 10-min immersion, and co-cultivation for 3 days yielded the highest frequency of transient gfp expression, shoots regeneration response and transformation efficiency. By applying these optimized parameters we recovered independent transformed plants at the transformation efficiency of 23.33% on selection medium (MT salts augmented with 0.5 mg l⁻¹ BA, 0.5 mg l⁻¹ KT, 0.1 mg l⁻¹ NAA, 50 mg l⁻¹ kanamycin and 250 mg l⁻¹ cefotaxime). Expression of gfp in the leaf segments and regenerated shoots was confirmed using fluorescence microscope. Polymerase chain reaction (PCR) analysis using gfp and nptII gene-specific primers further confirmed the integration of the transgene in the independent transgenic plants. The transformation methodology described here may pave the way for generating transgenic plants using leaf segments as explants.     

Simulated microgravity affects semicarbazide-sensitive amine oxidase expression in recombinant Escherichia coli by HPLC-ESI-QQQ analysis

Simulated microgravity has been reported to affect the gene, protein expression, and its function in the cells. Semicarbazide-sensitive amine oxidase (SSAO; E.C.1.4.3.6.) is widely distributed in vascular cells, smooth muscle cells, and adipocytes. It is noteworthy whether the expression of SSAO is affected under simulated microgravity or not. In this study, an SSAO-transformed Escherichia coli BL21 was constructed firstly. Then, a sensitive, selective, and accurate method based on high-performance liquid chromatography electrospray ionization triple quadrupole (HPLC-ESI-QQQ) was developed to determine the amount of SSAO in the E. coli BL21. The limit of detection and limit of quantification were 5.0 and 10 fmol, respectively. Finally, SSAO expression in the recombinant E. coli BL21 was evaluated with various gravity and temperature conditions by HPLC-ESI-QQQ analysis. It is interesting that the tendency in the alteration of SSAO under simulated microgravity showed temperature difference. At 18 °C, the amount of SSAO in the inclusion bodies and soluble fractions under the simulated microgravity increased by 83% and 116%, respectively, compared with normal gravity. However, the decrease by 38% and 49% in the inclusion bodies and soluble fractions under the simulated microgravity was observed at 37 °C. Results obtained here indicate that the SSAO expression under simulated microgravity is dramatically sensitive to the temperature. On the other hand, a novel bioreactor from this study may also be useful for the recombinant protein expression in the field of gene engineering.     

Clinical relevance of cyclooxygenase-2 and matrix metalloproteinases (MMP-2 and MT1-MMP) in human breast cancer tissue

Breast cancer (BC) is the most common neoplasm among women in most developed countries, including Egypt. Elevated levels of certain proteins in human BC are associated with unfavorable prognosis and progressive stages of the disease. The aim of our study was to evaluate the protein expression profile and prognostic significance of cyclooxygenase-2 (COX-2), matrix metalloproteinase-2 (MMP-2), MMP-9 and membrane type 1-MMP (MT1-MMP) and their interaction in operable BC patients. The protein expression of COX-2, MMP-2 and MT1-MMP were evaluated by western blot technique, whereas enzymatic activity of MMP-2 and MMP-9 was determined by zymography in 47 breast cancer patients as well as normal adjacent tissues. Also, the correlation between these proteins and age, tumor size, LN stage, TNM stage, estrogen receptor, progesterone receptor, disease-free survival, and overall survival (OS) has been investigated. As compared to adjacent normal tissues, COX-2, MMP-2 and MT1-MMP were over-expressed in 43, 64, and 60 % of tumor tissues, respectively. In the same pattern, the activity of MMP-2 (62 %) and MMP-9 (45 %) was elevated in BC tissues. Multivariate analysis showed a positive correlation between the protein expression of COX-2, MMP-2, and MT1-MMP and the activity of MMP-2 and MMP-9 in BC patients. However, the enzymatic activity showed no correlation with clinicopathological features. This study confirms the preclinical evidence that COX-2 increased the expression of MT1-MMP, which in turn activates MMP-2. The lack of correlation with clinicopathological features, OS or disease-free survival ascertains the complexity of tumor progression and metastasis with many pro- and counter regulatory factors.     

Synthesis and antibacterial activity of substituted flavones, 4-thioflavones and 4-iminoflavones

Synthesis of flavones, 4-thioflavones and 4-iminoflavones was carried out with the substitution of variable halogens, methyl, methoxy and nitro groups in the A, B and AB rings of the respective compounds and we also report here their antibacterial activity. Most of the synthesized compounds were found to be active against Escherichia coli, Bacillus subtilis, Shigella flexnari, Salmonella aureus, Salmonella typhi and Pseudomonas aeruginosa. Activity of 4-thioflavones and 4-iminoflavones was found to be higher than that of their corresponding flavone analogues. Investigated compounds having substituents like F, OMe and NO2 at 4'-position in ring-B exhibited enhanced activity and the presence of electronegative groups in the studied compounds showed a direct relationship to the antibacterial activity.     

Physiological and biochemical changes in plants under waterlogging

Waterlogging usually results from overuse and/or poor management of irrigation water and is a serious constraint with damaging effects. The rapidly depleting oxygen from submerged root zone is sensed and plant adjusts expressing anaerobic proteins. Plant cells shift their metabolism towards low energy yielding anaerobic fermentation pathways in the absence of oxygen. Structural modifications are also induced as aerenchyma formation and adventitious rootings, etc. Studies at molecular and biochemical levels to facilitate early perception and subsequent responses have also been worked out to produce resistant transgenic plants. This review explores the sequential changes of plant responses at different levels regarding their defense strategies and efforts made to enhance them, tailoring crucial regulators so that they can withstand waterlogging stress.     

Prophylactic Treatment with a G Glycoprotein Monoclonal Antibody Reduces Pulmonary Inflammation in Respiratory Syncytial Virus (RSV)-Challenged Na?ve and Formalin-Inactivated RSV-Immunized BALB/c Mice

We examined whether prophylactically administered anti-respiratory syncytial virus (anti-RSV) G monoclonal antibody (MAb) would decrease the pulmonary inflammation associated with primary RSV infection and formalin-inactivated RSV (FI-RSV)-enhanced disease in mice. MAb 131-2G administration 1 day prior to primary infection reduced the pulmonary inflammatory response and the level of RSV replication. Further, intact or F(ab′)₂ forms of MAb 131-2G administered 1 day prior to infection in FI-RSV-vaccinated mice reduced enhanced inflammation and disease. This study shows that an anti-RSV G protein MAb might provide prophylaxis against both primary infection and FI-RSV-associated enhanced disease. It is possible that antibodies with similar reactivities might prevent enhanced disease and improve the safety of nonlive virus vaccines.Respiratory syncytial virus (RSV) infection in infants and young children causes substantial bronchiolitis and pneumonia (11, 27, 28, 40) resulting in 40,000 to 125,000 hospitalizations in the United States each year (27). RSV is also a prominent cause of respiratory illness in older children; those of any age with compromised cardiac, pulmonary, or immune systems; and the elderly (6, 7, 11, 17, 18, 39). Despite extensive efforts toward vaccine development (3, 5, 8, 20, 30, 38), none is yet available. Currently, only preventive measures are available that focus on infection control to decrease transmission and prophylactic administration of a humanized IgG monoclonal antibody (MAb) directed against the F protein of RSV (palivizumab) that is recommended for high-risk infants and young children (4, 7, 17). To date, no treatment has been highly effective for active RSV infection (17, 21).The first candidate vaccine, a formalin-inactivated RSV (FI-RSV) vaccine developed in the 1960s, not only failed to protect against disease but led to severe RSV-associated lower respiratory tract infection in young vaccine recipients upon subsequent natural infection (8, 16). The experience with FI-RSV has limited nonlive RSV vaccine development for the RSV-naïve infant and young child. Understanding the factors contributing to disease pathogenesis and FI-RSV vaccine-enhanced disease may identify ways to prevent such a response and to help achieve a safe and effective vaccine.The RSV G, or attachment, protein has been implicated in the pathogenesis of disease after primary infection and FI-RSV-enhanced disease (2, 26, 31). The central conserved region of the G protein contains four evolutionarily conserved cysteines in a cysteine noose structure, within which lies a CX3C chemokine motif (9, 29, 34). The G protein CX3C motif is also immunoactive, as suggested by studies with the mouse model that show that G protein CX3C motif interaction with CX3CR1 alters pulmonary inflammation (41), RSV-specific T-cell responses (12), FI-RSV vaccine-enhanced disease, and expression of the neurokinin substance P (14) and also depresses respiratory rates (32). Recent studies demonstrated that therapeutic treatment with a murine anti-RSV G protein monoclonal antibody (MAb 131-2G) which blocks binding to CX3CR1 can reduce pulmonary inflammation associated with primary infection (13, 23). These findings led us to hypothesize that prophylactic administration of this anti-RSV G monoclonal antibody may also diminish pulmonary inflammation associated with RSV infection in naïve and in FI-RSV-vaccinated mice. In this study, we evaluate the impact of prophylactic administration of MAb 131-2G on the pulmonary inflammatory response to primary infection and to RSV challenge following FI-RSV immunization in mice.     

Synthesis and anti-inflammatory activity of some selected aminothiophene analogs

Some 2-aminothiophene analogs 1-6 were synthesized and characterized. Among the tested compounds, compound 1 (IC50 121.47 microM) exhibited highest while the compound 5 showed least anti-inflammatory potential (IC50 422 microM).