Anti-liver cancer activity of TNF-related apoptosis-inducing ligand gene and its bystander effects

AIM:To observe the anti-liver cancer activity of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) gene and its bystander effects on hepatocellular carcinoma (HCC) cell line SMMC7721.METHODS:Full-length cDNA of human TRAIL was transferred into SMMC7721 cells with a binary adenoviral vector system.Polymerase-chain reaction following reverse transcription (RT-PCR) was used to determine the expression of TRAIL gene. Effects of the transfected gene on proliferation of SMMC7721 cells were measured by MTT assay. Its influence on apoptosis was demonstrated by fluorescence-activated cell sorting (FACS). The bystander effect was observed by co-culturing the SMMC7721 cells with and without the transfected TRAIL gene at different ratios, and the culture medium supernatant from the transfected cells was also examined for its influence on SMMC7721 cells.RESULTS:The growth-inhibition rate and apoptotic cell fraction in the cells transfected with the TRAIL gene, Bax gene or only LacZ gene were 91.2%, 48.0%, 28.8% and 29.1%, 12.5%, 6.6%, respectively. The growth-inhibition rate of transfection with these three sequences in normal human fibroblasts was 6.1%, 45.5% and 7.6%, respectively,indicating a discriminative inhibition of TRAIL transfection on the cancer cells. In the co-culturing test, addition of the transfected TRAIL to SMMC7721 cells in proportions of 5%,25%, 50%, 75% and 100%, resulted in a growth-inhibition of 15.9%, 67%, 80.2%, 86.4% and 87.7%, respectively.We failed to observe a significant growth-inhibition effect of the culture medium supernatant on SMMC7721 cells.CONCLUSION: TRAIL gene transferred by a binary adenoviral vector system can inhibit proliferation of SMMC7721 cellsand induce their apoptosis. A bystander effect was observed,which seemed not to be mediated by soluble factors.

A series of aminoamides used for flotation of kaolinite

N-(2-aminoethyl)-dodecanamide, N-(3-dimethylaminopropyl)-dodecanamide, and N-(3-diethylaminoproyl)-dodecanamide used as collectors were studied for the flotation of kaolinite in the absence of additives at different pulp pHs as well as different collector contents. The effectiveness of the long chain aminoamides on pure kaolinite was demonstrated in laboratory scale flotation tests. The adsorption mechanism of the aminoamides onto kaolinite was investigated through zeta potential determinations and infrared spectrometry. The -98 μm size fractions of kaolinite, taken from Jiaxian Henan of China, were used in flotation. The hydrophilic group size of the aminoamides has a relatively less influence upon the floatability of pure kaolinite. The results suggest either the static-electric force or the coordinating bond adsorption mechanism of the aminoamides onto kaolinite depends on pulp pH.

Effects of cholesterol on proliferation and functional protein expression in rabbit bile duct fibroblasts

AIM:To investigate the effect of cholesterol (Ch) on the growth and functional protein expression of rabbit bile duct fibroblasts.METHODS:The cultured bile duct fibroblasts were divided randomly into two groups:the control group and the experiment group (fibroblasts were incubated respectively with 0.6g/L Ch for 12, 24, 36 and 48 h).The growth and DNA synthesis of bile duct fibroblasts were measured by the means of ^3H-TdR incorporation.The total protein content of fibroblast was measured by BSA protein assay reagent kit,then the expression of α-actin was analyzed semiquantitatively by Western blot.RESULTS:After treatment with 0.6g/L Ch for 12, 24,36 and 48h, the values of ^3H-TdR incorporation of bile duct fibroblasts were respectively 3.1±0.39, 3.8±0.37,4.6±0.48 and 5.2±0.56mBq/cell, and the values of the corresponding control groups were 3.0±0.33, 3.2±0.39,3.7±0.49 and 4.3±0.43mBq/cell. After comparing the values of experiment groups and their corresponding control groups,it was found that the 3H-TdR incorporation of bile duct fibroblasts after treatment with 0.6g/L Ch for 24, 36 and 48h were significantly increased (P<0.05, P<0.01, P<0.01),while the ^3H-TdR incorporation of 12-h group was not different statistically from its control group.Ch had no obvious effect on the total protein content of fibroblasts.After incubated with 0.6g/L Ch for 12, 24, 36 and 48h,the total protein content of each experiment group was not altered markedly compared with its corresponding control group.The values of experiment groups were 0.246±0.051,0.280±0.049, 0.263±0.044 and 0.275±0.056ng/cell, and those of corresponding control groups were 0.253±0.048,0.270±0.042,0.258±0.050 and 0.270±0.045 ng/cell.Western blot analysis revealed that the α-actin expression in fibroblasts affected by Ch for 12 and 24 h was not markedly changed compared with their corresponding control groups (P>0.05),the values of total gray scale of 12- and 24-h groups were 1 748±185 and 1 756±173,respectively. But after stimulation with Ch for 36 h, the total gray scale of fibroblasts (1923±204) was significantly higher than that of control group (1734±197).When the time of Ch treatment was lengthened to 48h, the α-actin expression was markedly elevated, the total gray scale was 2189±231 (P<0.01 vs control group).CONCLUSION:Moderately concentrated Ch can promote the proliferation of bile duct fibroblasts at early stage, With the prolongation of Ch treatment, the α-actin expression of fibroblasts was also increased,but the hypertrophy of fibroblasts was not observed,

Healing of hydrogen-attacked cracks in split specimens with recovering heat treatment in vacuum

The healing mechanism of hydrogen-attacked cracks in low carbon steel andCr-Mo steel and its influencing factors during the healing process were studied by recovering heattreatment of split specimens in vacuum. The result showed that crack pacing turns much smaller underthe condition of pure heating, especially for crack tips. The healing effect is well related to thelength of cracks with the shorter in priority. By the primary mechanism of thermal diffusion, ironand carbon atoms must diffuse at the high speed in steel to realize that plasticity deformationenergy exceeds and overcomes surface tensile force energy. In addition, phase transformation andstress-stain relationship also have positive effects on the process.

A Genome Sequence of Novel SARS-CoV Isolates: the Genotype, GD-Ins29, Leads to a Hypothesis of Viral Transmission in South China

We report a complete genomic sequence of rare isolates (minor genotype) of the SARS-CoV from SARS patients in Guangdong, China, where the first few cases emerged. The most striking discovery from the isolate is an extra 29-nucleotide sequence located at the nucleotide positions between 27,863 and 27,864 (referred to the complete sequence of BJ01) within an overlapped region composed of BGI-PUP5 (BGI-postulated uncharacterized protein 5) and BGI-PUP6 upstream of the N (nucleocapsid) protein. The discovery of this minor genotype, GD-Ins29, suggests a significant genetic event and differentiates it from the previously reported genotype, the dominant form among all sequenced SARS-CoV isolates. A 17-nt segment of this extra sequence is identical to a segment of the same size in two human mRNA sequences that may interfere with viral genome replication and transcription in the cytosol of the infected cells. It provides a new avenue for the exploration of the virus-host interaction in viral evolution, host pathogenesis, and vaccine development.

The E Protein Is a Multifunctional Membrane Protein of SARS-CoV

The E (envelope) protein is the smallest structural protein in all coronaviruses and is the only viral structural protein in which no variation has been detected. We conducted genome sequencing and phylogenetic analyses of SARS-CoV. Based on genome sequencing, we predicted the E protein is a transmembrane (TM) protein characterized by a TM region with strong hydrophobicity and α-helix conformation. We identified a segment (NH2-_L-Cys-A-Y-Cys-Cys-N_-COOH) in the carboxyl-terminal region of the E protein that appears to form three disulfide bonds with another segment of corresponding cysteines in the carboxyl-terminus of the S (spike) protein. These bonds point to a possible structural association between the E and S proteins. Our phylogenetic analyses of the E protein sequences in all published coronaviruses place SARS-CoV in an independent group in Coronaviridae and suggest a non-human animal origin.

Site-specific determination of TTR-related functional peptides by using scanning tunneling microscopy

For the design and optimization of functional peptides, unravelling the structures of individual building blocks as well as the properties of the ensemble is paramount. TI＇R1, derived from human transthyretin, is a fibril-forming peptide implicated in diseases such as familial amyloid polyneuropathy and senile systemic amyloidosis. The functional peptide TTR1-RGD, based on a TFR1 scaffold, was designed to specifically interact with cells. Here, we used scanning tunneling microscopy （STM） to analyze the assembly structures of TTRl-related peptides with both the reverse sequence and the modified forward sequence. The site- specific analyses show the following： i） The TIR1 peptide is involved in assembly, nearly covering the entire length within the ordered [3-sheet structures, ii） For TTR1-RGD peptide assemblies, the TTR1 motif forms the ordered [3-sheet while the RGDS motif adopts a flexible conformation allowing it to promote cell adhesion. The key site is clearly identified as the linker residue Gly13. iii） Close inspection of the forward and reverse peptide assemblies show that in spite of the difference in chemistry, they display similar assembling characteristics, illustrating the robust nature of these peptides, iv） Glycine linker residues are included in the ^-strands, which strongly suggests that the sequence could be optimized by adding more linker residues. These garnered insights into the assembled structures of these peptides help unravel the mechanism driving peptide assemblies and instruct the rational design and optimization of sequence- programmed peptide architectures.

Complete Genome Sequences of the SARS-CoV: the BJ Group (Isolates BJ01-BJ04)

Beijing has been one of the epicenters attacked most severely by the SARS-CoV (severe acute respiratory syndrome-associated coronavirus) since the first patient was diagnosed in one of the city's hospitals. We now report complete genome sequences of the BJ Group, including four isolates (Isolates BJ01, BJ02, BJ03, and BJ04) of the SARS-CoV.It is remarkable that all members of the BJ Group share a common haplotype, consisting of seven loci that differentiate the group from other isolates published to date. Among 42 substitutions uniquely identified from the BJ group, 32 are non-synonymous changes at the amino acid level. Rooted phylogenetic trees, proposed on the basis of haplotypes and other sequence variations of SARS-CoV isolates from Canada, USA, Singapore, and China, gave rise to different paradigms but positioned the BJ Group, together with the newly discovered GD01 (GD-Ins29) in the same clade, followed by the H-U Group (from Hong Kong to USA) and the H-T Group (from Hong Kong to Toronto), leaving the SP Group (Singapore) more distant. This result appears to suggest a possible transmission path from Guangdong to Beijing/Hong Kong, then to other countries and regions.

Genome Organization of the SARS-CoV

Annotation of the genome sequence of the SARS-CoV (severe acute respiratory syndrome-associated coronavirus) is indispensable to understand its evolution and pathogenesis. We have performed a full annotation of the SARS-CoV genome sequences by using annotation programs publicly available or developed by ourselves. Totally, 21 open reading frames (ORFs) of genes or putative uncharacterized proteins (PUPs) were predicted. Seven PUPs had not been reported previously, and two of them were predicted to contain transmembrane regions. Eight ORFs partially overlapped with or embedded into those of known genes, revealing that the SARS-CoV genome is a small and compact one with overlapped coding regions. The most striking discovery is that an ORF locates on the minus strand. We have also annotated non-coding regions and identified the transcription regulating sequences (TRS) in the intergenic regions. The analysis of TRS supports the minus strand extending transcription mechanism of coronavirus. The SNP analysis of different isolates reveals that mutations of the sequences do not affect the prediction results of ORFs.

Evolution and Variation of the SARS-CoV Genome

Knowledge of the evolution of pathogens is of great medical and biological significance to the prevention, diagnosis, and therapy of infectious diseases. In order to understand the origin and evolution of the SARS-CoV (severe acute respiratory syndrome-associated coronavirus), we collected complete genome sequences of all viruses available in GenBank, and made comparative analyses with the SARS-CoV. Genomic signature analysis demonstrates that the coronaviruses all take the TGTT as their richest tetranucleotide except the SARS-CoV. A detailed analysis of the forty-two complete SARS-CoV genome sequences revealed the existence of two distinct genotypes, and showed that these isolates could be classified into four groups. Our manual analysis of the BLASTN results demonstrates that the HE (hemagglutinin-esterase) gene exists in the SARS-CoV, and many mutations made it unfamiliar to us.

The R Protein of SARS-CoV: Analyses of Structure and Function Based on Four Complete Genome Sequences of Isolates BJ01-BJ04

The R (replicase) protein is the uniquely defined non-structural protein (NSP) responsible for RNA replication, mutation rate or fidelity, regulation of transcription in coronaviruses and many other ssRNA viruses. Based on our complete genome sequences of four isolates (BJ01-BJ04) of SARS-CoV from Beijing, China, we analyzed the structure and predicted functions of the R protein in comparison with 13 other isolates of SARS-CoV and 6 other coronaviruses. The entire ORF (open-reading frame) encodes for two major enzyme activities, RNA-dependent RNA polymerase (RdRp) and proteinase activities. The R polyprotein undergoes a complex proteolytic process to produce 15 function-related peptides. A hydrophobic domain (HOD) and a hydrophilic domain (HID) are newly identified within NSP1. The substitution rate of the R protein is close to the average of the SARS-CoV genome. The functional domains in all NSPs of the R protein give different phylogenetic results that suggest their different mutation rate under selective pressure. Eleven highly conserved regions in RdRp and twelve cleavage sites by 3CLP (chymotrypsin-like protein) have been identified as potential drug targets. Findings suggest that it is possible to obtain information about the phy-logeny of SARS-CoV, as well as potential tools for drug design, genotyping and diagnostics of SARS.