Synthesis and characterization of cisplatin-loaded, EGFR-targeted biopolymer and in vitro evaluation for targeted delivery.

The design of smart targeted drug delivery systems that deliver drugs to specific cancer cells will give rise to cancer treatments with better efficacy and lower toxicity levels. We report the development and characterizations of maleimide-functionalized biopolymer (Mal-PGA-Asp) as an effective targeted drug delivery carrier synthesized from an amidation reaction between aspartylated PGA (PGA-Asp) and N-(maleimidohexanoyl)-ethylenediamine (NME). The epidermal growth factor receptor (EGFR) targeting peptide (TP13) was conjugated to Mal-PGA-Asp to obtain the targeting carrier (TP13-Mal-PGA-Asp). Cisplatin was finally loaded by complexation to form a biocompatible and tumor targeted therapeutic drug (TP13-Mal-PGA-Asp3-Pt). The resultant biopolymer with an average size 87 ± 28 nm showed a sustainable release profile with a half-maximal release time (t(1/2)) of approximately 15 h in physiological saline. Fluorescence imaging and flow cytometry analysis revealed that TP13 significantly enhanced the cellular uptake of TP13-Mal-PGA-Asp3-Pt in the human hepatoma cell line SMMC-7721. The IC(50) value demonstrated the superior anticancer activity of TP13-Mal-PGA-Asp3-Pt over PGA-Asp-Pt. Therefore, the newly developed drug carrier (TP13-Mal-PGA-Asp) obtained in this study may provide an efficient and targeted delivery of anticancer drugs, presenting a promising targeted chemotherapy in EGFR-positive cancers.

Protective effect of recombinant human glucagon-like peptide-1 (rhGLP-1) pretreatment in STZ-induced diabetic mice.

Human glucagon-like peptide-1 (hGLP-1) and its mimetics have emerged as therapies for type 2 diabetes. However, clinical treatment of diabetes with hGLP-1 is ineffective because of rapid DPPIV-mediated hGLP-1 degradation in the circulation. In this study, we investigated the protective effect of recombinant human glucagon-like peptide-1 (rhGLP-1) treatment on STZ-induced diabetic mice. Mice were treated daily with rhGLP-1 (24 nmol/kg body weight) starting before or after STZ injection (40 mg/kg body weight) to induce diabetes. Mice pretreated with rhGLP-1 before but not after STZ showed significantly reduced blood glucose levels (P < 0.05), increased oral glucose tolerance (area under the curve, 1740 ± 71.18 vs 2416 ± 205.6, P < 0.05). Furthermore, the bioproduct of lipid peroxidation, MDA, was reduced and SOD and GSH-PX activities were enhanced globally and in pancreas of mice that received rhGLP-1 pretreatment before STZ, when comparing with STZ-treated mice. Finally, STZ-induced pancreatic islet damage was rescued by rhGLP-1 pretreatment. Taken together, the results of this study demonstrate that rhGLP-1 pretreatment has a protective effect against STZ-induced diabetes in mice. These findings suggest that the GLP-1 pretreatment may be a new therapeutic strategy in the preventive and protective treatment during diabetes initiation and progression.

Identification of differentially expressed genes induced by Bamboo mosaic virus infection in Nicotiana benthamiana by cDNA-amplified fragment length polymorphism.

BACKGROUND: The genes of plants can be up- or down-regulated during viral infection to influence the replication of viruses. Identification of these differentially expressed genes could shed light on the defense systems employed by plants and the mechanisms involved in the adaption of viruses to plant cells. Differential gene expression in Nicotiana benthamiana plants in response to infection with Bamboo mosaic virus (BaMV) was revealed using cDNA-amplified fragment length polymorphism (AFLP). RESULTS: Following inoculation with BaMV, N. benthamiana displayed differential gene expression in response to the infection. Isolation, cloning, and sequencing analysis using cDNA-AFLP furnished 90 cDNA fragments with eight pairs of selective primers. Fifteen randomly selected genes were used for a combined virus-induced gene silencing (VIGS) knockdown experiment, using BaMV infection to investigate the roles played by these genes during viral infection, specifically addressing the means by which these genes influence the accumulation of BaMV protein. Nine of the 15 genes showed either a positive or a negative influence on the accumulation of BaMV protein. Six knockdown plants showed an increase in the accumulation of BaMV, suggesting that they played a role in the resistance to viral infection, while three plants showed a reduction in coat protein, indicating a positive influence on the accumulation of BaMV in plants. An interesting observation was that eight of the nine plants showing an increase in BaMV coat protein were associated with cell rescue, defense, death, aging, signal transduction, and energy production. CONCLUSIONS: This study reports an efficient and straightforward method for the identification of host genes involved in viral infection. We succeeded in establishing a cDNA-AFLP system to help track changes in gene expression patterns in N. benthamiana plants when infected with BaMV. The combination of both DNA-AFLP and VIGS methodologies made it possible to screen a large number of genes and identify those associated with infections of plant viruses. In this report, 9 of the 15 analyzed genes exhibited either a positive or a negative influence on the accumulation of BaMV in N. benthamiana plants.

Addition of a cysteine to glucagon-like peptide-1 (GLP-1) conjugates GLP-1 to albumin in serum and prolongs GLP-1 action in vivo.

Glucagon-like peptide-1 (GLP-1) is a promising new therapeutic agent for the treatment of type 2 diabetes. However, GLP-1 has a short half-life (t(1/)(2)<2min) due to rapid degradation by dipeptidyl peptidase IV in vivo. To circumvent this problem, a recombinant mGLP-1 with a cysteine at the C-terminus of GLP-1 was expressed in Escherichia coli and purified by affinity and reverse-phase chromatography. This addition of a cysteine facilitates mGLP-1 binding to serum albumin both in vitro and in vivo, thus protecting mGLP-1 from protease degradation. Similar to GLP-1, mGLP-1 stimulated cAMP production in PC12 cells and exhibited insulinotropic activity in MIN6 cells under in vitro culture conditions. Importantly, in glucose tolerance tests mice treated with mGLP-1 exhibited much lower glucose levels and much higher insulin levels versus that in mice treated with unmodified GLP-1. Furthermore, the effects of mGLP-1 on reduction of blood glucose levels lasted for 6-7h, while the effects of unmodified GLP-1 only lasted for 0.5-1h after injection. These results demonstrate that mGLP-1 is biologically active and its pharmaceutical efficacy is largely enhanced by the cysteine-mediated covalent conjugation with albumin in the serum after injection. Therefore, the mGLP-1 with a cysteine may be a better potential therapeutic drug than the unmodified GLP-1 for treating type 2 diabetes.

Commensal bacteria regulate Toll-like receptor 3-dependent inflammation after skin injury.

The normal microflora of the skin includes staphylococcal species that will induce inflammation when present below the dermis but are tolerated on the epidermal surface without initiating inflammation. Here we reveal a previously unknown mechanism by which a product of staphylococci inhibits skin inflammation. This inhibition is mediated by staphylococcal lipoteichoic acid (LTA) and acts selectively on keratinocytes triggered through Toll-like receptor 3(TLR3). We show that TLR3 activation is required for normal inflammation after injury and that keratinocytes require TLR3 to respond to RNA from damaged cells with the release of inflammatory cytokines. Staphylococcal LTA inhibits both inflammatory cytokine release from keratinocytes and inflammation triggered by injury through a TLR2-dependent mechanism. To our knowledge, these findings show for the first time that the skin epithelium requires TLR3 for normal inflammation after wounding and that the microflora can modulate specific cutaneous inflammatory responses.

Detection of mutant p53 protein in workers occupationally exposed to benzidine.

To investigate the expression of mutant p53 protein in workers occupationally exposed to benzidine, we detected mutant p53 protein by immuno-PCR assay in the serum of 331 benzidine-exposed healthy workers, while we classified exfoliated urothelial cells in urine samples with Papanicoloau's grading (PG). The Papanicoloau's grading classified exfoliated urothelial cells of the subjects from grade I (normal cells) to grade III (suspicious malignant cells). The subjects were also divided into high, medium and low exposure groups according to the exposure intensity index. The results revealed that mutant p53 protein in the medium and high exposure groups were significantly higher than the in low exposure group (p<0.05), and in PG II and III were significantly higher than in the PG I (p<0.05). There was no significant differences among Papanicoloau's gradings strata in the low exposure group on the incidence and quantity of mutant p53 protein. In the medium and high exposure groups, the incidence and/or quantity of mutant p53 protein in the stratum of PG II and/or III were significantly higher than that of PG I (p<0.05). Detection of mutant p53 protein in conjunction with benzidine exposure level and Papanicoloau's gradings of exfoliated urothelial cells could provide more information to help us elevate surveillance efficiency and diagnose bladder cancer in the early period.

[Preparation and biological activity of poly (gamma-glutamic acid) -cisplatin conjugate].

Preparation of a poly (gamma-glutamic acid)-cisplatin conjugate was introduced and its in vitro antitumor effect was investigated. Poly (gamma-glutamic acids) was obtained by using fermentation methods. The hydrolyzed small molecular weight of poly (gamma-glutamic acids) was prepared by acid hydrolysis. The interaction between poly (gamma-glutamic acids) -cisplatin conjugate (PGA-CDDP) and DNA was investigated by PCR model. MTT assay was used to investigate the in vitro anticancer activity of the conjugate. Apoptosis assay of the conjugate was investigated by FCM assay and the in vivo toxicity was also proceeded. The results showed that the poly (gamma-glutamic acids) -cisplatin conjugate was obtained successfully and its yield is 10% - 12%. It has obvious antitumor effects on human liver tumor BEL7404 cells, human lung tumor H446 cells and human colon tumor RKO cells. At the same time, it also has apoptosis effects on the three kinds of tumor cell lines. The in vivo toxicity of PGA-CDDP was examined in normal mice and the results showed that the in vivo toxicity of this conjugate was significantly lower than that of free CDDP. In conclusion, the poly (gamma-glutamic acids) -cisplatin conjuate could be used as a potential clinic antitumor drug. The poly (gamma-glutamic acids) obtained by fermentation can be used as a valuable drug carrier system.

Functional and structural characterization of recombinant dermcidin-1L, a human antimicrobial peptide.

Antimicrobial peptides from human skin are an important component of the innate immune response and play a key role as a first line of defense against infections. One such peptide is the recently discovered dermcidin-1L. To better understand its mechanism and to further investigate its antimicrobial spectrum, recombinant dermcidin-1L was expressed in Escherichia coli as a fusion protein and purified by affinity chromatography. The fusion protein was cleaved by factor Xa protease to produce recombinant dermcidin-1L. Antimicrobial and hemolytic assays demonstrated that dermcidin-1L displayed microbicidal activity against several opportunistic nosocomial pathogens, but no hemolytic activity against human erythrocytes even at concentrations up to 100 microM. Structural studies performed by circular dichroism spectroscopy indicated that the secondary structure of dermcidin-1L was very flexible, and both alpha-helix and beta-sheet structures might be required for the antimicrobial activity. Our results confirmed previous findings indicating that dermcidin-1L could have promising therapeutic potentials and shed new light on the structure-function relationship of dermcidin-1L.

[The use of gene chip in detecting Mycobacterium tuberculosis resistant to rifampin and isoniazid].

OBJECTIVE: To study the application of gene chip in detecting Mycobacterium tuberculosis resistant to rifampin (RFP) and isoniazid (INH). METHODS: Probes were designed and the gene chip was fabricated according to the 30 single nucleotide polymorphisms of 11 mutations on 4 genes associated with RFP and INH resistance. The mutations in Mycobacterium tuberculosis were detected by gene chip to analyze the resistance to INH and RFP. RESULTS: 85 of 110 (77.3%) strains resistant to INH and 22 of 30 (73.3%) strains sensitive to INH were detected, while 77 of 94 (81.9%) strains resistant to RFP and 40 of 46 (87.0%) strains sensitive to RFP were detected. The results from the gene-chip detection were consistent with the sequence information. CONCLUSION: The gene-chip technology, a fast test with high accuracy, specificity and sensitivity, as shown in our study, is promising in the clinical detection of Mycobacterium tuberculosis resistant to INH and RFP.

A new approach to random mutagenesis in vitro.

Random mutagenesis is a powerful tool for studying the effects of a large number of permutations of a particular DNA sequence and its encoded products. Here we describe a new strategy of conducting in vitro random mutagenesis using ethyl methane sulfonate (EMS). The Bacillus aprN18 gene, coding for a serine protease with fibrinolytic activity, was used as a target gene. To study the mutations of the coding region, rather than the whole plasmid, the 1.4 kb gene fragment was cut out from an expression plasmid and treated with 10 mM EMS at 37 degrees C for 1 h. The treated fragment was then ligated back into the original expression vector and a library of random mutants was constructed in a protease-deficient Bacillus subtilis strain. A plate assay-based screening method was used to select for mutant clones with altered enzyme activity, and the change of activity was then confirmed by a semi-quantitative enzyme assay using liquid culture supernatant. The inserts of five clones with altered enzyme activity were randomly chosen for sequencing analysis. Among the point mutations detected, GC --> AT transition accounts for 42.1%, AT --> GC transition 34.2% and GC/CG transversion 23.7%, respectively. To our knowledge this is the first application of EMS for in vitro mutagenesis of a defined DNA sequence.

[Screening and identification of egg mimotope of Schistosoma japonicum].

OBJECTIVE: To explore egg mimotope of Schistosoma japonicum that can be used in the development of diagnostic reagents for schistosomiasis. METHODS: By performing three rounds of biopanning in the affinity selection before picking out single clones for identification, target specific phages were effectively enriched from a random 15-peptides phage library with an immobilized mAb 6B12 which is specific to egg antigen of S. japonicum as a bait. By using ELISA, competitive ELISA with natural egg antigen as competitor, and Western blotting, 13 phage clones with high affinity and specificity to 6B12 were obtained and amplified from 400 single clones. RESULTS: DNA sequencing revealed that all the 13 selected single clones were identical in displaying gene sequence. Serum samples were tested by ELISA for the presence of IgG antibodies to the phages with mimic epitope, and showed that there was a significant difference between healthy volunteers and schistosomiasis patients. CONCLUSION: It is possible that the phage display peptide antigen, egg mimotope of S. japonicum may be a replacement of natural egg antigen for the diagnosis of schistosomiasis.