The high transmissibility and genetic variability of the influenza virus have made the design of effective approaches to control the infection particularly challenging. The virus surface hemagglutinin (HA) protein is responsible for the viral attachment to the host cell surface via the binding with its glycoligands, such as sialyllactose (SL), and thereby is an attractive target for antiviral designs. Herein we present the facile construction and development of two SL-incorporated chitosan-based materials, either as a water-soluble polymer or as a functional fiber, to demonstrate their abilities for viral adhesion inhibition and decontamination. The syntheses were accomplished by grafting a lactoside bearing an aldehyde-functionalized aglycone to the amino groups of chitosan or chitosan fiber followed by the enzymatic sialylation with sialyltransferase. The obtained water-soluble SL-chitosan conjugate bound HA with high affinity and inhibited effectively the viral attachment to host erythrocytes. Moreover, the SL-functionalized chitosan fiber efficiently removed the virus from an aqueous medium. The results collectively demonstrate that these potential new materials may function as the virus adsorbents for prevention and control of influenza. Importantly, these materials represent an appealing approach for presenting a protein ligand on a chitosan backbone, which is a versatile molecular platform for biofunctionalization and, thereby, can be used for not only antiviral designs, but also extensive medical development such as diagnosis and drug delivery.
Objective: We have previously shown GM3 to positively regulate TNF-α expression via a PI3K/Akt pathway in mouse melanoma B16 cells [Wang et al.: Biochem Biophys Res Commun 2007;356:438–443]. The GM3 signal was shown to be located upstream of Akt, but whether it is located upstream of PI3K and which molecule is the effector of PI3K remain to be clarified. Methods: We used inhibitors of PI3K and mTOR, and siRNA directed to Rictor, Raptor and Rho-GDP dissociation inhibitor β (Arhgdib). Results: PI3K inhibitors LY294002 and LY303511 were shown to suppress TNF-α expression that is stimulated by GM3 in B16 cells, suggesting that the GM3 signal is located upstream of the PI3K-Akt pathway. Rapamycin suppressed TNF-α expression, indicating mTOR to be involved in the pathway. Either siRNA Raptor or siRNA Rictor suppressed TNF-α expression, but the latter suppressed the effects of GM3 on TNF-α expression and Akt phosphorylation at Ser473, indicating the GM3 signal to be transduced via Rictor/mTOR and Akt (Ser473), leading to TNF-α stimulation. Finally, Arhgdib, the tumor suppressor gene whose expression is associated with GM3, was shown to be upstream of TNF-α. Conclusions: The GM3 signal is thus transduced in B16 cells through a PI3K, Rictor/mTOR, Akt, Arhgdib pathway, leading to stimulated expression ofTNF-α.
Ly-GDI, Rho GTPase dissociation inhibitor beta, was found to be expressed parallel to the GM3 level in mouse B16 cells whose GM3 contents were modified by B4galt6 sense, B4galt6 antisense cDNA, or St3galt5 siRNA transfection. Ly-GDI expression was increased on GM3 addition to these cells and decreased with D-PDMP treatment, a glucosylceramide synthesis inhibitor. Suppression of GM3 or Ly-GDI by RNAi was concomitantly associated with an increase in anchorage-independent growth in soft agar. These results clearly indicate that GM3 suppresses anchorage-independent growth through Ly-GDI. GM3 signals regulating Ly-GDI expression was inhibited by LY294002, siRNA against Akt1 and Akt2 and rapamycin, showing that GM3 signals are transduced via the PI3K/Akt/ mTOR pathway. Either siRNA towards Rictor or Raptor suppressed Ly-GDI expression. The Raptor siRNA suppressed the effects of GM3 on Ly-GDI expression and Akt phosphorylation at Thr 308 , suggesting GM3 signals to be transduced to mTOR-Raptor and AktThr 308 , leading to Ly-GDI stimulation. siRNA targeting Pdpk1 reduced Akt phosphorylation at Thr 308 and rendered the cells insensitive to GM3 stimulation, indicating that Akt-Thr 308 plays a critical role in the pathway. The components aligned in this pathway showed similar effects on anchorage-independent growth as GM3 and Ly-GDI. Taken together, GM3 signals are transduced in B16 cells through PI3K, Pdpk1, Akt Thr308 and the mTOR/Raptor pathway, leading to enhanced expression of Ly-GDI mRNA, which in turn suppresses anchorage-independent growth in melanoma B16 cells. (Cancer Sci 2011; 102: 1476-1485 L y-GDI, a Rho GTPase dissociation inhibitor beta, is also known as RhoGDI2 or D4-GDI. It belongs to a family of Rho GDP dissociation inhibitors (RhoGDI) including RhoGDI1 and RhoGDI3. The family is named for its ability to inhibit the dissociation of bound GDP from its partner Rho GTPase, which regulates interactions with regulatory guanine nucleotide exchange factors, GTPase-activating proteins, and effector targets. (1) Rho GTPases, such as RhoA, Rac1 and CDC42, are important regulators of cellular migration and adhesion, (2,3) and regulate the dynamics of b-actin, resulting in stress fibers, lamellipodia, and filopodia formation. In contrast to other GDIs, Ly-GDI interacts poorly with RhoA, Rac and Cdc42 in vivo. (4) Although Ly-GDI differs substantially at the structural level from the other two GDIs, RhoGDI-1 and RhoGDI-3, these three GDI exhibit very similar biological regulatory activities on Rho family proteins. (5-7) Ly-GDI is regarded as an invasion and metastasis suppressor gene in human bladder cancer cells. (8,9) For example, the truncated Ly-GDI has been shown to promote metastasis of colon cancer. (10) However, Ly-GDI was reported to be highly expressed in human breast cancer cells and its involvement in tumor cell invasion has been suggested. (11) Thus, function of Ly-GDI is controversial, and furthermore, it remains unknown as to how Ly-GDI is regulated in cells and how its regulation is related to tumor be...
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