Qiaozhen Kang

Anti-tumor effects of a novel chimeric peptide on S180 and H22 xenografts bearing nude mice.

In recent years, many endogenous peptides have been identified by screening combinatory phage display peptide library, which play important roles in the process of angiogenesis. A heptapeptide, ATWLPPR, binds specifically to NRP-1 and selectively inhibits VEGF165 binding to VEGFR-2. Another heptapeptide, NLLMAAS, blocks both Ang-1 and Ang-2 binding to Tie-2 in a dose-dependent manner. In the present study, we aimed to connect ATWLPPR (V1) with NLLMAAS (V2) via a flexible linker, Ala-Ala, to reconstruct a novel peptide ATWLPPRAANLLMAAS (V3). We firstly investigated the anti-tumor and anti-angiogenic effects of peptide V3 on sarcoma S180 and hepatoma H22 bearing BALB/c nude mice. Mice were continuously subcutaneously administrated with normal saline, V1 (320microg/kg/d), V2 (320microg/kg/d), V1+V2 (320microg/kg/d), and V3 (160, 320 and 480microg/kg/d), for 7 days. Treatment with peptide V3 could significantly reduce the tumor weight and volume. Pathological examination showed that the tumors treated with peptide V3 had a larger region of necrosis than that of peptide V1, V2, and V1+V2 at the same dose. A significant decrease of microvessel density (MVD) in a dose-dependent manner was observed in each group of peptide V3. The results of pathological examination on normal tissue, lung, heart, liver, spleen, kidney and white blood cells showed that peptide V3 might have no significant toxicity. In conclusion, our results demonstrated that peptide V3 could be more effective on inhibiting tumor growth and angiogenesis than that of V1, V2, and V1+V2. Peptide V3 could be considered as a novel chimeric peptide with potent anti-tumor activity.

A Golgi-associated protein 4.1B variant is required for assimilation of proteins in the membrane

The archetypal membrane skeleton is that of the erythrocyte, consisting predominantly of spectrin, actin, ankyrin R and protein 4.1R. The presence in the Golgi of a membrane skeleton with a similar structure has been inferred, based on the identification of Golgi-associated spectrin and ankyrin. It has long been assumed that a Golgi-specific protein 4.1 must also exist, but it has not previously been found. We demonstrate here that a hitherto unknown form of protein 4.1, a 200 kDa 4.1B, is associated with the Golgi of Madin-Darby canine kidney (MDCK) and human bronchial epithelial (HBE) cells. This 4.1B variant behaves like a Golgi marker after treatment with Brefeldin A and during mitosis. Depletion of the protein in HBE cells by siRNA resulted in disruption of the Golgi structure and failure of Na+/K+-ATPase, ZO-1 and ZO-2 to migrate to the membrane. Thus, this newly identified Golgi-specific protein 4.1 appears to have an essential role in maintaining the structure of the Golgi and in assembly of a subset of membrane proteins.

Cytoskeletal protein 4.1R negatively regulates T-cell activation by inhibiting the phosphorylation of LAT

Protein 4.1R (4.1R) was first identified in red cells where it plays an important role in maintaining mechanical stability of red cell membrane. 4.1R has also been shown to be expressed in T cells, but its function has been unclear. In the present study, we use 4.1R-deficient mice to explore the role of 4.1R in T cells. We show that 4.1R is recruited to the immunologic synapse after T cell-antigen receptor (TCR) stimulation. We show further that CD4+ T cells of 4.1R-/- mice are hyperactivated and that they displayed hyperproliferation and increased production of interleukin-2 (IL-2) and interferon gamma (IFNgamma). The hyperactivation results from enhanced phosphorylation of LAT and its downstream signaling molecule ERK. The 4.1R exerts its effect by binding directly to LAT, and thereby inhibiting its phosphorylation by ZAP-70. Moreover, mice deficient in 4.1R display an elevated humoral response to immunization with T cell-dependent antigen. Thus, we have defined a hitherto unrecognized role for 4.1R in negatively regulating T-cell activation by modulating intracellular signal transduction.

Antitumor activity of novel chimeric peptides derived from cyclinD/CDK4 and the protein transduction domain 4

CyclinD1/CDK4 and cyclinD3/CDK4 complexes are key regulators of the cell progression and therefore constitute promising targets for the design of anticancer agents. In the present study, the key peptide motifs were selected from these two complexes. Chimeric peptides with these peptides conjugated to the protein transduction domain 4 (PTD4) were designed and synthesized. The chimeric peptides, PTD4-D1, PTD4-D3, PTD4-K4 exhibited significant anti-proliferation effects on cancer cell lines. These peptides could compete with the cyclinD/CDK4 complex and induce the G1/S phase arrest and apoptosis of cancer cells. In the tumor challenge experiment, these peptides showed potent antitumor effects with no significant side effects. Our results suggested that these peptides could be served as novel leading compounds with potent antitumor activity.

The membrane-cytoskeletal protein 4.1N is involved in the process of cell adhesion, migration and invasion of breast cancer cells

Protein 4.1N belongs to the protein 4.1 superfamily that links transmembrane proteins to the actin cytoskeleton. Recent evidence has shown that protein 4.1 is important in tumor suppression. However, the functions of 4.1N in the metastasis of breast cancer are largely unknown. In the present study, MCF-7, T-47D and MDA-MB-231 breast cancer cell lines with various metastatic abilities were employed. Protein 4.1N was found to be expressed in poorly metastatic MCF-7 and middle metastatic T-47D cell lines, and was predominantly associated with cell-cell junctions. However, no 4.1N expression was detected in the highly metastatic MDA-MB-231 cells. Moreover, re-expression of 4.1N in MDA-MB-231 cells inhibited cell adhesion, migration and invasion. The results suggest that protein 4.1N is a negative regulator of cell metastasis in breast cancer.

A Recombinant DNA Plasmid Encoding the sIL-4R-NAP Fusion Protein Suppress Airway Inflammation in an OVA-Induced Mouse Model of Asthma

Asthma is a chronic inflammatory airway disease. It was prevalently perceived that Th2 cells played the crucial role in asthma pathogenesis, which has been identified as the important target for anti-asthma therapy. The soluble IL-4 receptor (sIL-4R), which is the decoy receptor for Th2 cytokine IL-4, has been reported to be effective in treating asthma in phase I/II clinical trail. To develop more efficacious anti-asthma agent, we attempt to test whether the Helicobacter pylori neutrophil-activating protein (HP-NAP), a novel TLR2 agonist, would enhance the efficacy of sIL-4R in anti-asthma therapy. In our work, we constructed a pcDNA3.1-sIL-4R-NAP plasmid, named PSN, encoding fusion protein of murine sIL-4R and HP-NAP. PSN significantly inhibited airway inflammation, decreased the serum OVA-specific IgE levels and remodeled the Th1/Th2 balance. Notably, PSN is more effective on anti-asthma therapy comparing with plasmid only expressing sIL-4R.

Protective effects of soybean protein and egg white protein on the antibacterial activity of nisin in the presence of trypsin

The using of nisin to prevent foodborne pathogens (Staphylococcus aureus and Listeria monocytogenes) from contamination has received broad attentions during meat processing. However, the application of nisin has been limited because its antibacterial activity may be inhibited by trypsin. In this study, the protective effects of soybean protein and egg white protein on antibacterial activity of nisin were evaluated. It could be concluded that exogenous trypsin decreased the antibacterial activity of nisin, soybean protein and egg white protein could keep the nisin activity from enzymolysis of trypsin. Trypsin inhibitors in soybean protein and egg white protein could protect the antibacterial activity of nisin. Nisin with soybean protein or egg white protein in cooked meat product presented better quality preservation effects than nisin alone in the presence of trypsin. The total viable counts (TVC) and total volatile basic nitrogen (TVB-N) of nisin-treated group were significantly higher than these in nisin-soybean protein-treated and nisin-egg white protein-treated groups with trypsin. This study showed the potential of using soybean protein and egg white protein to stabilize the antibacterial activity of nisin under high trypsin conditions.

Purification, characterization and biological activities of a polysaccharide from Lepidium meyenii leaves

The characteristics and biological activities of a novel polysaccharide from Lepidium meyenii leaves (LMLP) were investigated. LMLP was purified using DEAE-52 cellulose chromatography followed by SephadexG-100 chromatography. The average molecule weight of LMLP was 58.43kDa and it was composed of galactose, arabinose, rhamnose, glucose and mannose with a relative molar ratio of 5.51:4.05:1.15:0.77:0.01. Antioxidant activity results showed that LMLP presented an EC50 of 3.72mg/mL in scavenging 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical, and it also had reducing power (OD700nm being 0.079 at 1mg/mL). Moreover, LMLP possessed the potential on stimulating immune response of RAW264.7 cells. It could promote proliferation, strengthen phagocytosis function, enhance the expression of CD80, and increase the secretion of nitric oxide (NO) in a dose-dependent manner. All of these results suggested that LMLP could be used as a natural ingredient for functional food.

Antitumor and immunomodulatory effects of recombinant fusion protein rMBP-NAP through TLR-2 dependent mechanism in tumor bearing mice.

The pro-inflammatory and immunomodulatory properties of Helicobacter pylori neutrophil activating protein (Hp-NAP) not only make it to play an important role in disease pathogenesis but also make it to be a potential candidate for therapeutic applications, including vaccine and drug development. Our previous work demonstrated that the recombinant Hp-NAP fused with the maltose binding protein of Escherichia coli (rMBP-NAP) play an important role in regulating the differentiation of Th1 cells. In this study, we investigated the ability of rMBP-NAP to induce antitumor immunity using two murine models of hepatoma H22 and sarcoma S180. Subcutaneous administration of mice with rMBP-NAP resulted in an about 40%-50% decrease of tumor growth compared with that of the control mice. Splenocytes from the tumor-bearing mice treated with rMBP-NAP showed a significant accumulation of CD4(+) IFN-γ-secreting cells, which is a cytokine profile of Th1 cells. Furthermore, intravenous injection of T2.5, toll like receptor (TLR) 2 blocking antibody, significantly recede the antitumor effect of rMBP-NAP and the production of IFN-γ induced by rMBP-NAP. Our findings indicate that potentiality of rMBP-NAP to be a candidate for the development of immunomodulatory antitumoral drugs.

Protein 4.1N is required for the formation of the lateral membrane domain in human bronchial epithelial cells

The membrane skeleton forms a scaffold on the cytoplasmic side of the plasma membrane. The erythrocyte membrane represents an archetype of such structural organization. It has been documented that a similar membrane skeleton also exits in the Golgi complex. It has been previously shown that βII spectrin and ankyrin G are localized at the lateral membrane of human bronchial epithelial cells. Here we show that protein 4.1N is also located at the lateral membrane where it associates E-cadherin, β-catenin and βII spectrin. Importantly, depletion of 4.1N by RNAi in human bronchial epithelial cells resulted in decreased height of lateral membrane, which was reversed following re-expression of mouse 4.1N. Furthermore, although the initial phase of lateral membrane biogenesis proceeded normally in 4.1N-depleted cells, the final height of the lateral membrane of 4.1N-depleted cells was shorter compared to that of control cells. Our findings together with previous findings imply that 4.1N, βII spectrin and ankyrin G are structural components of the lateral membrane skeleton and that this skeleton plays an essential role in the assembly of a fully functional lateral membrane.