Urinary measurement of circulating tumor DNA for treatment monitoring and prognosis of metastatic colorectal cancer patients.

Background Solid tumor tissue testing is the gold standard for molecular-based assays for metastatic colorectal cancer (mCRC). This poses challenges during treatment monitoring. Total DNA derived from urine specimens offers clear advantages to track the disease dynamics. Our study aims to evaluate the sensitivity for total DNA recovered from urine and its clinical relevance to mCRC. Methods KRAS mutations in urine specimens were examined in 150 mCRC patients. Baseline concordance was established to determined clinical relevance. The total DNA quantities were also prospectively examined in serial samplings during treatment. Results Analysis of the genetic mutations showed good agreement for baseline samples. Matched tumor and urine specimens' molecular profiles were observed to have 90% concordance. Comparing with healthy volunteers, we established a cutoff of 8.15 ng that demonstrated elevated total DNA levels was associated with mCRC patients (sensitivity: 90.7%; specificity: 82.0%). For patients treated with chemotherapy or anti-epidermal growth factor receptor inhibitors, DNA quantity mirrored early treatment response. Survival analysis showed that patients with sustained elevated quantities of KRAS mutations had poorer outcome. Conclusions Total urine DNA offers a viable complement for mutation profiling in mCRC patients, given the good agreement with matched tumor samples. Our study also established that this is specific based on the results from healthy individuals. Serial monitoring of total DNA levels allowed early prediction to treatment response and was effective to identify high risk patients. This is potentially useful to complement current disease management.

Preparation of PGA-PAE-Micelles for Enhanced Antitumor Efficacy of Cisplatin.

Poly-γ-l-glutamic acid (PGA) is an outstanding drug carrier candidate owning to its excellent biodegradability and biocompatibility. The PGA carrier may shield toxic drugs from the body and enable the delivery of poorly soluble or unstable drugs and thereby minimize the side effects and improve drug efficacy. However, the limitation of PGA as a drug carrier is low drug loading efficiency (DLE), which is usually below 30%. In this study, we reported a chemical modification method using l-phenylalanine ethyl ester (PAE). PGA-PAE construct was amphiphilic, which could form micelles in aqueous solution. Cisplatin (CDDP), a commonly used chemotherapy drug whose side effect is well-known, was used as a model molecule to test the drug-loading efficiency of PGA-PAE. In this paper, two sizes of CDDP-loaded PGA-PAE micelles (M(Pt)-1 and M(Pt)-2) were prepared, the average diameter of M(Pt)-1 was 106 ± 6 nm and M(Pt)-2 was 210 ± 9 nm. The DLE of M(Pt)-1 and M(Pt)-2 was 52.8 ± 2.2 and 55.8 ± 1.2%, respectively. Both exhibited excellent biocompatibility, stability, and drug-retaining capability in physiological condition. The in vitro accumulative drug-releasing profile, IC50 for different tumor cell lines HeLa, A549, and HCCC9810, and in vivo pharmacokinetics were similar between these two micelles; however, M(Pt)-1 showed higher tumor tissue retention and longer efficient cancer cell internalization time (up to 20 d). Our results suggested PGA-PAE micelle carriers reduced the toxicity of CDDP and its size at around 100 nm was the better for CDDP high-efficacy.

A novel GLP-1 analog, a dimer of GLP-1 via covalent linkage by a lysine, prolongs the action of GLP-1 in the treatment of type 2 diabetes.

GLP-1 is an incretin hormone that can effectively lower blood glucose, however, the short time of biological activity and the side effect limit its therapeutic application. Many methods have been tried to optimize GLP-1 to extend its in vivo half-time, reduce its side effect and enhance its activity. Here we have chosen the idea to dimerize GLP-1 with a C-terminal lysine to form a new GLP-1 analog, DLG3312. We have explored the structure and the biological property of DLG3312, and the results indicated that DLG3312 not only remained the ability to activate the GLP-1R, but also strongly stimulated Min6 cell to secrete insulin. The in vivo bioactivities have been tested on two kinds of animal models, the STZ induced T2DM mice and the db/db mice, respectively. DLG3312 showed potent anti-diabetic ability in glucose tolerance assay and single-dose administration of DLG3312 could lower blood glucose for at least 10 hours. Long-term treatment with DLG3312 can reduce fasted blood glucose, decrease water consumption and food intake and significantly reduce the HbA1c level by 1.80% and 2.37% on STZ induced T2DM mice and the db/db mice, respectively. We also compared DLG3312 with liraglutide to investigate its integrated control of the type 2 diabetes. The results indicated that DLG3312 almost has the same effect as liraglutide but with a much simpler preparation process. In conclusion, we, by using C-terminal lysine as a linker, have synthesized a novel GLP-1 analog, DLG3312. With simplified preparation and improved physiological characterizations, DLG3312 could be considered as a promising candidate for the type 2 diabetes therapy.

Expression and purification of Canis interferon α in Escherichia coli using different tags.

The potent and broad activity of Canis interferon α (CaIFNα) makes it an attractive candidate for the treatment of many viral diseases of dogs. Here, we fused CaIFNα to three different protein tags: thioredoxin (Trx), glutathione S-transferase (GST), and NusA (Nus), to facilitate its expression and purification in Escherichia coli. The Trx-CaIFNα and GST-CaIFNα fusion proteins formed inclusion bodies, while the Nus-CaIFNα protein was soluble when expressed at low temperatures. Trx-CaIFNα was purified from inclusion bodies and refolded, while Nus-CaIFNα was purified under native conditions. The purity of Trx-CaIFNα and Nus-CaIFNα was greater than 90%, and their yields were 74.8% and 6.5%, respectively. Both Trx-CaIFNα and Nus-CaIFNα had antiviral activity in vitro. Their anti-viral activity was 1.09±0.47×10(14) and 2.25±0.87×10(12) U/mol, respectively, on Madin-Darby canine kidney cells. Both purification methods had advantages and disadvantages. A greater amount of Trx-CaIFNα was obtained, but refolding was required to obtain active protein. In contrast, soluble Nus-CaIFNα did not require refolding, which saved time and materials. However, Nus-CaIFNα, which contained a larger tag, had lower activity than Trx-CaIFNα. In general, we provided two protocols to obtain large amounts of CaIFNα with high antiviral activity. These protocols may promote the clinical development of CaIFNα in treating viral diseases in dog.

Exploring the diversity of Acinetobacter populations in river water with genus-specific primers and probes.

This study aimed to explore the diversity of river water Acinetobacter populations using culture-dependent and -independent methods. Pyrosequencing indicated that 1.5% of the total sequences from Qiandeng River water were classified as Acinetobacter. Twelve Acinetobacter strains were isolated from three different sampling sites of the Qiandeng River. Based on culture-dependent methods, A. johnsonii, A. lwoffii and A. guillouiae were the most abundantly represented Acinetobacter strains among the upper, middle and downstream populations of the river. Probing of three Acinetobacter-enriched 16S rRNA gene libraries with the Acinetobacter specific probe Act660F revealed 42 unique 16S rRNA gene sequences exhibiting a similarity of 94.9-99.9% with the known Acinetobacter strains. Among the uncultured Acinetobacter sequences, 50%, 58.3% and 68.8% of those obtained from upstream sampling site A, middle stream sampling site B and downstream sampling site C were phylogenetically located within Group I. This Group represented the most abundant strains of Acinetobacter populations in river water based on culture-independent methods. The results indicated that culture-independent methods provide more detailed information on the diversity of Acinetobacter populations than that based on culture-dependent methods. Therefore, the development of new and efficient isolation methods to identify uncultured Acinetobacter species is required.

Virus-induced gene complementation reveals a transcription factor network in modulation of tomato fruit ripening.

Plant virus technology, in particular virus-induced gene silencing, is a widely used reverse- and forward-genetics tool in plant functional genomics. However the potential of virus technology to express genes to induce phenotypes or to complement mutants in order to understand the function of plant genes is not well documented. Here we exploit Potato virus X as a tool for virus-induced gene complementation (VIGC). Using VIGC in tomato, we demonstrated that ectopic viral expression of LeMADS-RIN, which encodes a MADS-box transcription factor (TF), resulted in functional complementation of the non-ripening rin mutant phenotype and caused fruits to ripen. Comparative gene expression analysis indicated that LeMADS-RIN up-regulated expression of the SBP-box (SQUAMOSA promoter binding protein-like) gene LeSPL-CNR, but down-regulated the expression of LeHB-1, an HD-Zip homeobox TF gene. Our data support the hypothesis that a transcriptional network may exist among key TFs in the modulation of fruit ripening in tomato.

Bacitracin-conjugated superparamagnetic iron oxide nanoparticles: synthesis, characterization and antibacterial activity.

Bacitracin-conjugated superparamagnetic iron oxide (Fe(3)O(4)) nanoparticles were prepared by click chemistry and their antibacterial activity was investigated. After functionalization with hydrophilic and biocompatible poly(acrylic acid), water-soluble Fe(3)O(4) nanoparticles were obtained. Propargylated Fe(3)O(4) nanoparticles were then synthesized by carbodiimide reaction of propargylamine with the carboxyl groups on the surface of the iron oxide nanoparticles. By further reaction with N(3)-bacitracin in a Cu(I)-catalyzed azide-alkyne cycloaddition, the magnetic Fe(3)O(4) nanoparticles were modified with the peptide bacitracin. The functionalized magnetic nanoparticles were characterized by powder X-ray diffraction, X-ray photoelectron spectroscopy, TEM, zeta-potential analysis, FTIR spectroscopy and vibrating-sample magnetometry. Cell cytotoxicity tests indicate that bacitracin-conjugated Fe(3)O(4) nanoparticles show very low cytotoxicity to human fibroblast cells, even at relatively high concentrations. In view of the antibacterial activity of bacitracin, the biofunctionalized Fe(3)O(4) nanoparticles exhibit an antibacterial effect against both Gram-positive and Gram-negative organisms, which is even higher than that of bacitracin itself. The enhanced antibacterial activity of the magnetic nanocomposites allows the dosage and the side effects of the antibiotic to be reduced. Due to the antibacterial effect and magnetism, the bacitracin-functionalized magnetic nanoparticles have potential application in magnetic-targeting biomedical applications.

Glucagon-like peptide-1(1-37) can enhance blood glucose homeostasis in mice.

Glucagon-like peptide-1 (GLP-1) is produced by the posttranslational processing of proglucagon and acts as a regulator of various homeostatic events. No blood glucose regulation role of GLP-1(1-37) has previously been identified. However, our findings in this study clearly showed that GLP-1(1-37) could lower blood glucose levels both in normal and diabetic mice. In vitro stability analysis demonstrated that GLP-1(1-37) was more stable than GLP-1(7-37), with 94.7% of the initial amount of peptide left after a 4h exposure to mouse serum. Moreover, GLP-1(1-37) was confirmed to be a highly potent agonist of the GLP-1 receptor (GLP-1R) by measuring the expression of the luciferase reporter gene expression in transiently transfected human embryonic kidney (HEK293) cells. Unlike the glucose lowering effect of GLP-1(7-37), the glucose-lowering effect of GLP-1(1-37) could not be blocked by the GLP-1R antagonist exendin(9-39), suggesting that GLP-1(1-37) might activate the GLP-1R via a different mechanism. Therefore, our findings suggest that GLP-1(1-37) could be a potential therapeutic drug for the treatment of type 2 diabetes in the future.

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.

A novel GAP460 biopolymer for use as a carrier in drug-delivery applications.

We synthesized a new non-toxic biopolymer (GAP460) containing γ,L-glutamic acid and aspartate (Asp). Conjugates of GAP460 and cisplatin exhibited a drug-carrying capacity of nearly 40%, 3-times higher than γ-PGA and dramatically decreasing the amount of biopolymer required for high-dose delivery. Treatment with GAP460-cisplatin conjugate (PACC) not only effectively inhibited tumor growth in nude mice, but also resulted in extended survival and lower nephrotoxicity, suggesting that GAP460 could be used as an effective carrier for drug delivery and that PACC may have potential therapeutic applications in the clinical treatment of cancer.

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.

Poly (γ, L-glutamic acid)-cisplatin bioconjugate exhibits potent antitumor activity with low toxicity: a comparative study with clinically used platinum derivatives.

We have recently synthesized a new platinum derivative, poly (γ, L-glutamic acid)-cisplatin conjugate (γ-PGA-CDDP), and shown that it displayed remarkable antitumor activity against breast tumor in a mouse model. The purpose of this study is to systematically compare this new drug with three platinum derivatives currently used in the clinic: cisplatin, carboplatin and oxaliplatin. Here, we show that γ-PGA-CDDP displays impressive antitumor activity over the current clinically used platinum drugs. More interestingly and more importantly, γ-PGA-CDDP conjugate significantly reduces cytotoxicity, mitigates oxidative stress and improves antioxidative capability in vivo. Animals treated with γ-PGA-CDDP display the same profile of body weight as the control animals, while the tumors in γ-PGA-CDDP-treated animals are significantly suppressed compared with those treated with carboplatin and oxaliplatin. Our data suggest that γ-PGA could be used as an effective carrier for drug delivery and that γ-PGA-CDDP conjugate may have potential therapeutic applications in human cancers that are sensitive to treatment with CDDP-based chemotherapy such as ovarian cancer.

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.

Effects of insulin-mimetic vanadyl-poly(gamma-glutamic acid) complex on diabetic rat model.

Poly-gamma-glutamic acid (gamma-PGA) prepared by fermentation of microbe was used as drug carrier for vanadium sulfate to obtain vanadyl-poly-gamma-glutamic acid (VO-gamma-PGA) complex. The FI-IR spectrum of the complex demonstrated that the expected VO-gamma-PGA complex is formed by the coordination of VO(2+) through the side chain carboxylic groups of the gamma-PGA. Studies of the complex in treating type I diabetes were carried out on alloxan induced diabetes rats. The results of treating the rats in 2 weeks and then stopping administration for 10 days showed that VO-gamma-PGA can effectively lower blood glucose levels of diabetic rats during administration. But after ceasing treatment there were no differences between groups in blood glucose level and water intake. The results of oral glucose tolerance and some serum parameters also demonstrated that VO-gamma-PGA was more effective than vanadium sulfate in treating diabetic rats.

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.

Kisspeptin-10, a KISS1-derived decapeptide, inhibits tumor angiogenesis by suppressing Sp1-mediated VEGF expression and FAK/Rho GTPase activation.

Kisspeptin-10 (Kp-10), a decapeptide derived from the primary translation product of KISS1 gene, has been reported previously to be a key hormone for puberty and an inhibitor for tumor metastasis via the activation of G protein-coupled receptor 54. However, whether Kp-10 inhibits angiogenesis, which is critical for tumor growth and metastasis and other human diseases, is still unknown. Here we show that Kp-10 significantly inhibits human umbilical vein endothelial cell (HUVEC) migration, invasion, and tube formation, key processes in angiogenesis. Using chicken chorioallantoic membrane assay and vascular endothelial growth factor (VEGF)-induced mouse corneal micropocket assay, we show that Kp-10 inhibits angiogenesis in vivo. Furthermore, Kp-10 inhibits tumor growth in severe combined immunodeficient mice xenografted with human prostate cancer cells (PC-3) through inhibiting tumor angiogenesis, whereas Kp-10 has little effect on the proliferation of HUVECs and human prostate cancer cells. In deciphering the underlying molecular mechanisms, we show that Kp-10 suppresses VEGF expression by inhibiting the binding of specificity protein 1 to VEGF promoter and by blocking the activation of c-Src/focal adhesion kinase and Rac/Cdc42 signaling pathways in HUVECs, leading to the inhibition of tumor angiogenesis.

A novel peptide from human apolipoprotein(a) inhibits angiogenesis and tumor growth by targeting c-Src phosphorylation in VEGF-induced human umbilical endothelial cells.

Many angiogenesis inhibitors are derived from large plasma proteins. Previous studies showed that the Kringle5-like domain (termed KV) in human apolipoprotein (a) is a potential antiangiogenic factor. However, its active region and the underling molecular mechanism remain elusive. Here, we identified an 11-amino acid peptide (named KV11) as the key region for the antiangiogenic function of the KV domain of apolipoprotein (a). We demonstrate that KV11 inhibits angiogenesis in vitro by suppressing human umbilical vein endothelial cell migration and microtubule formation. KV11 inhibits angiogenesis in chicken chorioallantoic membrane assays and mouse corneal micropocket angiogenesis assays in vivo. KV11 peptide shows no effect on tumor cell growth or proliferation, but significantly inhibits tumor growth in SCID mouse xenograft tumor model (p < 0.01) by preventing tumor angiogenesis. We elucidate that KV11 peptide suppresses angiogenesis and tumor progression by targeting the c-Src/ERK signaling pathways. Together, these studies provide the first evidence that KV11 from apolipoprotein KV domain has anti-angiogenesis functions and may be an anti-tumor drug candidate.

A single amino acid change in a geminiviral Rep protein differentiates between triggering a plant defence response and initiating viral DNA replication.

We have devised an in planta system for functional analysis of the replication-associated protein (Rep) of African cassava mosaic virus (ACMV). Using this assay and PCR-based random mutagenesis, we have identified an ACMV Rep mutant that failed to trigger the hypersensitive response (HR), but had an enhanced ability to initiate DNA replication. The mutant Rep-green fluorescent protein (GFP) fusion protein was localized to the nucleus. Sequence analysis showed that the mutated Rep gene had three nucleotide changes (A6-->T, T375-->G and G852-->A); only the A6-->T transversion resulted in an amino acid substitution (Arg to Ser), which is at the second residue in the 358 amino acid ACMV Rep protein. Our results indicate that a single amino acid can alter the differential ability of ACMV Rep to trigger the host-mediated HR defence mechanism and to initiate viral DNA replication. The implications of this finding are discussed in the context of plant-virus interactions.

Mutated recombinant human glucagon-like peptide-1 protects SH-SY5Y cells from apoptosis induced by amyloid-beta peptide (1-42).

Accumulation and deposition of amyloid beta peptide (Abeta) in the brain causes neuronal apoptosis and eventually leads to Alzheimer's disease (AD). A therapeutic target for AD is to block the cascade reaction induced by Abeta. It has been demonstrated that glucagon-like peptide-1 (GLP-1), which is an endogenous insulinotropic peptide secreted from the gut, binds to its receptor in the brain and possesses neuroprotective effects. Using site-directed mutagenesis and gene recombination techniques, we generated a mutated recombinant human glucagon-like peptide-1 (mGLP-1) which has longer half-life as compared with native GLP-1. This present work aims to examine whether mGLP-1 attenuates Abeta(1-42)-mediated cytotoxicity in SH-SY5Y cells and to explore the possible mechanisms. Our data indicate that > or = 0.02 ng/ml of mGLP-1 facilitated cell proliferation and 0.1 ng/ml and 0.5 ng/ml of mGLP-1 rescued SH-SY5Y cells from Abeta(1-42)-induced apoptosis. Moreover, Abeta(1-42) treatment dramatically stimulated the release of Ca(2+) from internal calcium stores in SH-SY5Y cells, while mGLP-1 helped to maintain the intracellular Ca(2+) homeostasis. Abeta(1-42) also significantly increased the expression level of TP53 and Bax genes which are involved in apoptotic pathways, and mGLP-1 decreased Abeta(1-42)-induced up-regulation of TP53 and Bax. Since mGLP-1 treatment elevated cytosolic cAMP concentration in SH-SY5Y cells, we postulate that mGLP-1 may exert its influence via binding to GLP-1 receptors in SH-SY5Y cells and stimulating the production of cAMP. These results suggest that mGLP-1 exhibited neuronal protection properties, and could potentially be a novel therapeutic agent for intervention in Alzheimer's disease.