Different cell kinetic changes in rat stomach cancer after treatment with celecoxib or indomethacin: Implications on chemoprevention

AIM: Mechanisms underlying the chemopreventive effects of cyclooxygenase (COX) inhibitors remain elusive. We have previously shown that celecoxib but not indomethacin could prevent carcinogen-induced gastric cancer development in Wistar rats. This chemopreventive effect appeared to be independent of COX-2 and prostaglandin (PG) E2 suppression since the lowest PGE2 was obtained in indomethacin group.This study compared the cell kinetic changes in stomachs of rats after treatment with celecoxib (5, 10, 20 mg/(kg·d)) or indomethacin (3 mg/(kg·d)) to gain more insights into the chemopreventive mechanism.METHODS: The apoptosis and proliferation indexes in gastric tumor, adjacent non-cancer tissues and normal gastric tissues were determined. Apoptosis was quantified by apoptotic nuclei counting and TUNEL, whereas proliferation was determined by Ki67 immunostaining.RESULTS: Treatment with either celecoxib or indomethacin inhibited gastric tumor proliferation by more than 65% (P＜0.02). However, celecoxib caused a dose-dependent increase in apoptosis (P＜0.05) which was not seen in indomethacin-treated tumors (P = 0.54). The highest apoptosis to proliferation ratio was seen in tumors treated with celecoxib at 10 mg/(kg·d). Treatment with this dose of celecoxib was associated with the lowest incidence of gastric cancer development.CONCLUSION: Our findings suggest that the difference in chemopreventive effects of indomethacin and celecoxib in this animal model of gastric carcinogenesis is largely due to the differential cell kinetic changes, which does not correlate with the degree of COX-2 and PG suppression.

Effects of cholesterol on the phenotype of rabbit bile duct fibroblasts

AIM: To investigate how cholesterol (Ch) can affect thephenotype of bile duct fibroblasts of New Zealand rabbits.METHODS: 16 rabbits were divided randomly into twogroups: the control group and the experiment group. Therabbits in experiment group were fed with hypercholesteroldiet for 8 weeks. Bile duct was dissociated from rabbits andprepared for transmission electron microscopy. The purifiedbile duct fibroblasts were cultured and divided randomlyinto there groups: control group, Ch smiddle concentrationgroup (0.6 g/L), Ch high concentration group (1.2 g/L). Afterincubated for 72 h, the fibroblasts were made into specimensfor transmission electron microscopy. The expression of α-actin in bile duct fibroblasts was measured by means oflaser scanning confocal microscopy.RESULTS: With the transmission electron microscopy, thenormal bile duct fibroblasts were shuttle-shaped, and therewere abundant rough endoplasmic reticulums (RER), butfew mitochondria or microfilaments in cytoplasm. This isthe typical phenotype of fibroblasts. Bile duct fibroblasts ofhypercholesterolemic rabbits were observed, by thetransmission electron microscopy Rough endoplasmicreticulums were significantly reduced, with a lot ofmicrofilament bundles or stress fibers appeared in cytoplasm,especially under plasma membrane. Dense bodies werescattered within these bundles. Macula densas anddiscontinuous sarcolemma were found under plasmamembrane. It suggested that the bile duct fibroblasts ofhypercholesterolemic rabbits presented the phenotype ofsmooth muscle cell. The cultured bile duct fibroblasts alsohad typical phenotype of fibroblasts. After stimulated bymiddle concentration cholesterol (0.6 g/L) for 72 h, thereappeared lots of microfilaments in cytoplasm, but withoutdense body, macula densa and discontinuous sarcolemma.Observed with confocal microscopy, there were many regularbundles of microfilaments in fibroblasts treated with middleconcentration ch (0.6 g/L) and the expression of α-actinwas signifiantly increased. The average fluorescence valueof middle concentration group was 1 628+189 (P＜0.01 vscontrol group). Microfilaments and the expression of α-actinwere greatly decreased in fibroblastes of high concentrationgroup (1.2 g/L). The average fluorescence value of highconcentration group was 1 427±153 (P＜0.05 vs middleconcentration group). There were a lower expression of α-actin and few microfilaments in bile duct fibroblasts of controlgroup with an average fluorescence value of 1 224±138.CONCLUSION: Cholesterol can make bile duct fibroblastshave the phenotypic characteristics of smooth muscle cellboth in vitro andin vivo and this effect is more significant invivo. The effect is probably associated with some otherfactors besides cholesterol.

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,

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 C-Terminal Portion of the Nucleocapsid Protein Demonstrates SARS-CoV Antigenicity

In order to develop clinical diagnostic tools for rapid detection of SARS-CoV (severe acute respiratory syndrome-associated coronavirus) and to identify candidate proteins for vaccine development, the C-terminal portion of the nucleocapsid (NC) gene was amplified using RT-PCR from the SARS-CoV genome, cloned into a yeast expression vector (pEGH), and expressed as a glutathione S-transferase (GST) and Hisx6 double-tagged fusion protein under the control of an inducible promoter. Western analysis on the purified protein confirmed the expression and purification of the NC fusion proteins from yeast. To determine its antigenicity, the fusion protein was challenged with serum samples from SARS patients and normal controls. The NC fusion protein demonstrated high antigenicity with high specificity, and therefore, it should have great potential in designing clinical diagnostic tools and provide useful information for vaccine development.

“Three Kingdoms” to Romance

For some historic reasons, our new journal is named 'Genomics, Pro teomics & Bioinformatics', or as we have nicknamed it in short the Journal of GPB. A growing number of '-ome' and '-omics' have appeared in many diverse fields of biology, especially in the recent years under profound influences of the Human Genome Project and many other genome projects completed or in progress. We had almost attempted to re-name this journal 'Ever-more-omics' to include all the new comers. However, after a second thought, we have decided to entertain these 'Three Kingdoms' first while we are keeping an eye on others.

Gene Identification and Expression Analysis of 86,136 Expressed Sequence Tags(EST)from the Rice Genome

Expressed Sequence Tag (EST) analysis has pioneered genome-wide gene discovery and expression profiling. In order to establish a gene expression index in the rice cultivar indica, we sequenced and analyzed 86,136 ESTs from nine rice cDNA libraries from the super hybrid cultivar LYP9 and its parental cultivars. We assembled these ESTs into 13,232 contigs and leave 8,976 singletons. Overall, 7,497 sequences were found similar to the existing sequences in GenBank and 14,711 are novel. These sequences are classified by molecular function, biological process and pathways according to the Gene Ontology. We compared our sequenced ESTs with the publicly available 95,000 ESTs from japonica, and found little sequence variation, despite the large difference between genome sequences. We then assembled the combined 173,000 rice ESTs for further analysis. Using the pooled ESTs, we compared gene expression in metabolism pathway between rice and Arabidopsis according to KEGG. We further profiled gene expression patterns in different tissues, developmental stages, and in a conditional sterile mutant, after checking the libraries are comparable by means of sequence coverage. We also identified some possible library specific genes and a number of enzymes and transcription factors that contribute to rice development.

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.

EST-based Analysis of Gene Expression in the Porcine Brain

Since pig is an important livestock species worldwide, its gene expressionhas been investigated intensively, but rarely in brain. In order to study gene expression profilesin the pig central nervous system, we sequenced and analyzed 43,122 high-quality 5'' end expressedsequence tags (ESTs) from porcine cerebellum, cortex cerebrum, and brain stem cDNA libraries,involving several different prenatal and postnatal developmental stages. The initial ESTs wereassembled into 16,101 clusters and compared to protein and nucleic acid databases in GenBank. Ofthese sequences, 30.6% clusters matched protein databases and represented function known sequences;75.1% had significant hits to nucleic acid databases and partial represented known function; 73.3%matched known porcine ESTs; and 21.5% had no matches to any known sequences in GenBank. We used thecategories defined by the Gene Ontology to survey gene expression in the porcine brain.

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.