Dissecting characteristics and dynamics of differentially expressed proteins during multistage carcinogenesis of human colorectal cancer

AIM: To discover novel biomarkers for early diagnosis, prognosis or treatment of human colorectal cancer. METHODS: i TRAQ 2D LC-MS/MS analysis was used to identify differentially expressed proteins(DEPs) in the human colonic epithelial carcinogenic process using laser capture microdissection-purified colonic epithelial cells from normal colon, adenoma, carcinoma in situ and invasive carcinoma tissues. RESULTS: A total of 326 DEPs were identified, and four DEPs(DMBT1, S100A9, Galectin-10, and S100A8) with progressive alteration in the carcinogenic process were further validated by immunohistochemistry. The DEPs were involved in multiple biological processes including cell cycle, cell adhesion, translation, m RNA processing, and protein synthesis. Some of the DEPs involved in cellular process such as "translation" and "m RNA splicing" were progressively up-regulated, while some DEPs involved in other processes such as "metabolism" and "cell response to stress" was progressively downregulated. Other proteins with up- or down-regulation at certain stages of carcinogenesis may play various roles at different stages of the colorectal carcinogenic process. CONCLUSION: These findings give insights into our understanding of the mechanisms of colorectal carcinogenesis and provide clues for further investigation of carcinogenesis and identification of biomarkers.

Extraction and identification of membrane proteins from black widow spider eggs

The eggs of oviparous animals are storehouses of maternal proteins required for embryonic development. Identification and molecular characterization of such proteins will provide much insight into the regulation of embryonic development. We previously analyzed soluble proteins in the eggs of the black widow spider （Latrodectus tredecimguttatus）, and report here on the extraction and mass spectrometric identification of the egg membrane proteins. Comparison of different lysis solutions indicated that the highest extraction of the membrane proteins was achieved with 3%-4% sodium laurate in 40 mmol/L Tris-HCI buffer containing 4% CHAPS and 2% DTT （pH 7.4）. SDS-PAGE combined with nLC- MS/MS identified 39 proteins with membranelocalization annotation, including those with structural, catalytic, and regulatory activities. Nearly half of the identified membrane proteins were metabolic enzymes involved in various cellular processes, particularly energy metabolism and biosynthesis, suggesting that relevant metabolic processes were active during the embryonic development of the eggs. Several identified cell membrane proteins were involved in the special structure formation and function of the egg cell membranes. The present proteomic analysis of the egg membrane proteins provides new insight into the molecular mechanisms of spider embryonic development.

A nuclear localized protein ZCCHC9 is expressed in cerebral cortex and suppresses the MAPK signal pathway

The CCHC-type zinc finger motif has numerous biological activities （such as DNA binding and RNA binding） and can also mediate protein-protein interaction. This article gives a primary report about the human ZCCHC9 gene. Protein ZCCHC9 contains four CCHC motifs and is highly conserved in humans, mice, and rats. The whole eDNA sequence of the ZCCHC9 gene has been amplified by PCR and a number of plasmids have been constructed for further study. The results show that ZCCHC9 is localized in the nucleus, and especially concentrated in the nueleolus. It is highly expressed in the brain and testicles of the mouse. This has been confirmed by real-time reverse transcription polymerase chain reaction （RT-PCR）. In situ hybridization of the mouse brain indicates that ZCCHC9 is mainly expressed in the cerebral cortex. Reporter gene assay shows that ZCCHC9 suppresses the transcription activities of NF-kappa B and SRE, and may play roles in the Mitogen-Aetivated Protein Kinase （MAPK） signaling transduetion pathway.

Influence of carbonyl stress on rheological alterations of blood materials and decarbonylation effect of glutathione

The effects of various toxic carbonyls such as malondialdehyde(MDA),a secondary product of lipid peroxidation,and other aldehydes on rheological parameters and their relationship with aging-associated alterations were studied.Both MDA and glutaraldehyde(Glu) in different concentrations significantly increase viscosity,plastic viscosity and yield stress of human plasma and erythrocyte suspensions.MDA(20 mmol/L) reduces sharply the typical fluorescence of proteins(excitation 280 nm/emission 350 nm),and produces age pigment-like fluorescence with a strong emission peak at 460 nm when excites at 395 nm by only being incubated for some hours.In contrast,Glu decreases merely the fluorescence of proteins without producing age pigment-like fluorescence.These data suggest interestingly that the MDA-induced gradual protein cross linking seems to form from different mechanisms compared to the fast rheological changes of blood materials which may take place either in acute and chronic diseases or during aging.On the other hand,MDA induces various deleterious alterations of erythrocytes whereas glutathione(GSH) inhibits the MDA-related carbonyl stress in a concentration-dependent manner.The results indicate that carbonyl-amino reaction exists in the blood widely and GSH has the ability to interrupt or reverse this reaction in a certain way.It implies that carbonyl stress may be one of the important factors in blood stasis and suggests a theoretical and practical approach in anti-stresses and anti-aging.

Expert systems for HPLC in biochemistry—The knowledge base for selection of HPLC column system in the separation of amino acids, peptides and proteins

The strategy of expert system for high performance liquid chromatography was discussed, the attentions are mainly placed on the knowledge base for selection of column system, separation modes and detection modes in the analysis of amino acids, peptides and proteins.

Structure Analysis of Streptococcal Protein G Fc Binding Domain

The gene fragment (191 bp) encoding protein G IgG Fc binding domain was isolated by PCR from group G streptococcus (CMCC32138), and a clone containing this gene fragment was found to give fine reactivity to human IgG when expressed in Escherichia coli. The complete nucleotide sequence of the gene fragment was determined. One base pair differs from previously reported protein Gnucleotide sequences, and resultsin an amino acid change (Ala-Thr), but this variation makes no difference in binding to the IgG Fc part by ELISA.The secondary structure of the protein G IgG Fc binding domain has been estimated by circular dichroism and assigned by computer algorithm.It shows a typical α-helix region in this domain.By breaking this α-helix region with recombinant DNA techniques, a 44 peptide, which contained the N-terminal 27 amino acid residues of this domain, was expressed in E. coli and showed no reactivity to IgG.The hydropathicity of this domain was also analyzed and compared with that of protein A relevant domain. Some similarity was found. These results suggest that the binding mechanism of protein G to the IgG Fc part depends on hydrophobic action which comes from the α-helix in protein G molecule, just as protein A binding to IgG Fc part.

Development and application of biological technologies in fish genetic breeding

Fish genetic breeding is a process that remolds heritable traits to obtain neotype and improved varieties.For the purpose of genetic improvement,researchers can select for desirable genetic traits,integrate a suite of traits from different donors,or alter the innate genetic traits of a species.These improved varieties have,in many cases,facilitated the development of the aquaculture industry by lowering costs and increasing both quality and yield.In this review,we present the pertinent literatures and summarize the biological bases and application of selection breeding technologies(containing traditional selective breeding,molecular marker-assisted breeding,genome-wide selective breeding and breeding by controlling single-sex groups),integration breeding technologies(containing cross breeding,nuclear transplantation,germline stem cells and germ cells transplantation,artificial gynogenesis,artificial androgenesis and polyploid breeding)and modification breeding technologies(represented by transgenic breeding)in fish genetic breeding.Additionally,we discuss the progress our laboratory has made in the field of chromosomal ploidy breeding of fish,including distant hybridization,gynogenesis,and androgenesis.Finally,we systematically summarize the research status and known problems associated with each technology.

Rapid growth and sterility of growth hormone gene transgenic triploid carp

Triploid carp(100%)with 150(3n=150)chromosomes were obtained by crossing the females of improved tetraploid hybrids(♀, 4n=200)of red crucian carp(♀)×common carp(♂)with the males of diploid yellow river carp(♂,2n=100).The crosses yielded transgenic triploid carp(positive triploid fish,44.2%of the progeny)and non-transgenic triploid carp(negative triploid fish). Histological examination of the gonads of 24-month-old positive triploid fish suggested they were sterile and the fish were not able to produce mature gametes during the breeding season.Morphologically,both the positive and negative triploid fish were similar.They had a spindle-shaped,laterally compressed,steel grey body with two pairs of barbells.Most of the quantifiable traits of the triploid carp were intermediate between those of the two parents.The positive and negative triploid fish were raised in the same pond for 2 years.The mean body weight of the positive triploid fish was 2.3 times higher than the negative triploid fish.The weight of the largest positive triploid fish was 2.91 times higher than that of the largest negative triploid fish.Thus,we produced fast-growing transgenic triploid carp that have a reduced ecological risk because of their inability to mate and produce progeny.

Evolutionary analysis of allotetraploid hybrids of red crucian carp×common carp,based on ISSR,AFLP molecular markers and cloning of cyclins genes

The allotetraploid hybrids of red crucian carp × common carp are the first reported artificially cultured polyploid fish with bisexual fertility and stable inheritance in vertebrate. Using ISSR and AFLP markers and the cyclins genes, the genomes and cyclin gene sequence changes were analyzed between the allotetraploid hybrids and their parents. The results indicated that the allotetraploids inherited many genetic characteristics from their parents and the genetic characteristics were stable after 15 generations. However, the allotetraploids had a closer genetic relationship with their original female parents and represented a bias toward the maternal progenitor. DNA fingerprinting analysis showed that the allotetraploids had undergone sequences deletion from their original parents and that the deleted sequences were mostly from the male parent's genome. Some non-parental bands were found in the allotetraploid hybrids. Sequences analysis of the cyclin A1 and B1 genes showed nonsynonymous substitutions of single nucleotides in codons that were different from their original parents, leading to non-parental amino acid loci. We speculate that the non-additivity in the allotetraploids, compared with their progenitors, could be an adjustment to the genomic shock from heterozygosity and polyploidy, allowing maintenance of genetic stability.

Genomic variation in the hybrids of white crucian carp and red crucian carp: evidence from ribosomal DNA

In this study, we conducted a cross of white crucian carp （♀）xred crucian carp （♂） （WR）, and characterized the morphology, reproduction and genetics of the progeny. Different from parents, WR with the gray color showed the hybrid morphological traits of both parents. WR possessed normal gonads producing mature eggs or sperm, and exhibited high fertilization rate （90.2%） and high hatchery rate （80.5%）, which contributed to produce and enlarge the population. WR with the same DNA content as parents was a diploid fish with 100 chromosomes （2n=100）. Amplified ITS of 45S rDNA, in WR the sequences consisting of 884 bp bases of the entire ITS-1 region, 5.8 S region, and entire ITS-2 region. The sequences showed high similarity between WR and its parents and leaned towards male inheritance. In WR, NTS of 5S rDNA consisted of three length types with total 654 bp bases. From sequence analysis of NTS, WR shared 94.2% and 95.1% similarities with their female and male parent, respectively. Sequence analysis of ITS and NTS revealed that there existed recombination and variation in the hybrid progeny, which was the genetic base for adaptation and speciation. In conclusion, we obtained WR from hybridization and it exhibited hybrid traits in morphology and variation in genetic composition showing essential difference with its parents. The obtainment of WR has important significance in fish genetic breeding.

The formation of improved tetraploid population of red crucian carp×common carp hybrids by androgenesis

Bisexual fertile diploid androgenetic individuals(A_(0))(2n=100)were formed by androgenesis.In this way,the diploid spermatozoa from male allotetraploid hybrids(AT)(4n=200)of red crucian carp(Carassius auratus red var.)(♀)×common carp(Cyprinus carpio L.)(♂)were used to fertilize the UV-treated hap-loid eggs of goldfish(Carassius auratus),and living androgenetic diploid fish were developed.The A_(0)became sexually mature at the age of 2 years,and they fertilized with each other to form their offspring(A_(1)).In this study,we observed the chromosomal number,gonadal structure and appearance of A_(1)fish.The results are as follows:(1)In A_(1),there were 85%tetraploids(A_(1)-4n),10%triploids(A_(1)-3n)and 5%diploids(A_(1)-2n),suggesting that diploid A_(0)could produce diploid gametes.It was concluded that the formation of diploid gametes generated from diploid A_(0)was probably related to the mechanism of pre-meiotic endoreduplication.(2)Among A_(1),only A_(1)-4n possessed normal ovaries and testes.The mature males of A_(1)-4n produced white semen.Under the electron microscope,the head of diploid sperm generated by A_(1)-4n was bigger than that of haploid sperm generated by red crucian carp.In the testes of the A_(1)-4n,there were many mature normal spermatozoa with a head bearing plasma mem-brane and a tail having the typical structure of"9+2"microtubules.Between the head and the tail,there were some mitochondria.The ovaries of A_(1)-4n developed well and mainly contained II,III and IV-stage oocytes.The IV-stage oocytes were surrounded by inner and outer follicular cells.The micropyle was observed on the oolemma of follicular cells.There were abundant yolks and plenty of endoplasmic reticulum in the cytoplasm of IV-stage oocytes.Because A_(1)-2n and A_(1)-3n were distant crossing diploid hybrids and triploid hybrids respectively,they possessed abnormal gonads,and no mature semen and eggs were observed.(3)Compared with allotetraploids,the A_(1)-4n fish not only had advantages such as fast growth rate and strong resistibility but also showed some new good performances such as high ratio of body width to body length,smaller heads and shorter tails.These results indicated that an-drogenesis could produce bisexual fertile tetraploids and improve the shape of allotetraploid hybrids as well,which will be of great significance in both the cell genetics research and fish breeding.

Comparative analysis of intermuscular bones in fish of different ploidies

We documented the number, morphology, and distribution of intermuscular bones in five fishes of different ploidy： Carassius auratus （Abbr.WCC, 2n=100）, Carassius auratus variety PengZe （Abbr.PZCC, 3n=150）, improved triploid crucian carp （Abbr.ITCC, 3n=150）, improved red crucian carp （Carassius auratus red var., Abbr.IRCC, , 2n=100）, and improved allotet- raploids （Abbr.GAT, （, 4n=200）. The number of intermuscular bones in WCC, PZCC, and GxAT ranged from 78 to 83 （n =81）, 80 to 86 （n =84）, and 77 to 84 （n=82）, respectively. The numbers in ITCC and IRCC were significantly lower, ranging from 77 to 82 （ n =79） and 58 to 77 （ n =71）, respectively. The average number of intermuscular bones in each sarco- mere, ranked in order from highest to lowest, was 0.721 （WCC）, 0.673 （PZCC）, 0.653 （GAT）, 0.633 （ITCC）, and 0.608 （IRCC）. There was no difference between ITCC and GxAT or between GxAT and PZCC. However, the average number of intermuscular bones in the sarcomeres of ITCC, WCC, and PZCC differed significantly, as did that of IRCC and the four other kinds of fish. The intermuscular bone of these five fishes was divided into seven shape categories, non-forked （I）, one-end-unequal-bi-fork （）, one-end-equal-bi-fork （Y）, one-end-multi-fork, two-end-bi-fork, two-end-multi-fork, and tree- branch types. Generally, the morphological complexity was higher in the anterior intermuscular bones than in the posterior body. The number of intermuscular bones was similar but not equal between the left and right sides of the body. ITCC had sig- nificantly fewer intermuscular bones than either WCC or PZCC, making it of greater commercial value. Additionally, IRCC and ITCC had fewer intermuscular bones than WCC. Our observations are significant in both fish bone developmental biology and genetic breeding.

Molecular cloning and differential expression patterns of the regulatory subunit B' gene of PP2A in goldfish,Carassius auratus

It is well established that the protein serine/threonine phosphatase 2A(PP2A) plays very important roles in many different cellular processes,including cell proliferation and differentiation,gene expression,neurotransmission,apoptosis,and aging.PP2A consists of three heterogenic subunits:the scaffold subunit A,the catalytic subunit C,and the regulatory subunit B.While both the scaffold and the catalytic subunits contain only two forms,at least four families of the regulatory subunits,B,B',B'',and B''' have been identified.These regulatory subunits from different families are encoded by different genes and bear other functions besides directing the specificity of PP2A.To study the functions of the regulatory subunits of PP2A in lower vertebrates,we have cloned the full-length cDNA sequence of the gene encoding the regulatory subunit B'δ of PP2A from gold fish,Carassius auratus using 3'-RACE and 5'-RACE cloning strategies.Our results revealed that the full-length B'δ cDNA contains 2415 bp and encodes a protein of 555 amino acids.The B'δ protein displays a very high level of sequence identity with the B'δ regulatory subunit from other species of vertebrates.Regarding its expression pattern,RT-PCR revealed that the highest level of mRNA was detected in brain,a less level detected in liver,spermary,ovary,kidney and gill,and the lowest level detected in the fin.During different developmental stages of gold fish,the highest level of mRNA expression was detected at the stages of two-cell,multiple-cell,blastula and gastrula,and a decreased level of B'δ mRNA was detected in other developmental stages.At the protein level,the highest expression level of B'δ protein was found in spermary,ovary,brain and heart,a less amount found in liver and the lowest level detected in kidney,gill and fin.Developmentally,B'δ protein was strongly expressed at the stages of two-cell,multiple-cell,blastula,gastrula,neurula,and optic vesicle,and then decreased at the stages of brain differentiation and eye pigmentation.These results suggest that B'δ appears to play a very important role during gold fish development and also in adult tissue homeostasis.

Black carp growth hormone gene transgenic allotetraploid hybrids of Carassius auratus red var. (♀)×Cyprinus carpio (♂)

Ecological safety is a major consideration in the commercialization of transgenic fish. Development of sterile transgenic triploid fish through hybridization of transgenic tetraploid fish and transgenic diploid fish is a feasible way to solve this problem. The "all-fish" transgene, pbcAbcGHc, containing the black carp β-actin gene promoter and the open reading frame (ORF) of the black carp growth hormone (GH) gene was constructed and introduced into fertilized eggs of allotetraploid fish through microinjection. Contrast cultivation results showed that the growth rate of 150 day-old P 0 black carp GH gene transgenic allotetraploid fish was much higher than that of controls. Sixty 150 day-old transgenic allotetraploid fish were assayed by PCR for transgene integration and 90% of fish were positive for the transgene. The transgene was detected in 13 of 20 sperm samples from male transgenic allotetraploid fish. RT-PCR detected transcription of the exogenous black carp GH gene in the muscle, liver, kidney and ovaries of the largest transgenic allotetraploid fish. This study has developed P 0 black carp GH gene transgenic allotetraploid fish with a highly increased growth rate, which provides a solid foundation for the establishment of a pure line of transgenic allotetraploid fish and for the large scale production of sterile transgenic triploid fish.

Pod1 is involved in the sexual differentiation and gonadal development of the Nile tilapia

Pod1 is a member of the basic helix-loop-helix(bHLH) family of transcription factors that have been implicated in the regulation of sexual differentiation and gonadal development in mammals.However,to date,little is known about the role of Pod1 in nonmammalian vertebrate gonadogenesis.We cloned and characterized the Pod1 gene from tilapia.The tilapia Pod1 gene contains an open reading frame(ORF) of 525 nucleotides which potentially codes for a protein with 174 amino acids.Sequence alignment revealed that the deduced tilapia protein sequence shared high homology(79.5% to 90.5%) with the Pod1 sequences of other vertebrates.The tissue distribution of Pod1 revealed by RT-PCR showed that it had varied expression patterns in adult tilapia.In situ hybridization was performed to examine the temporal and spatial expression patterns of Pod1 during tilapia sexual differentiation and gonadal development.In the undifferentiated gonad,Pod1 was expressed in the somatic cells of both sexes.Subsequently,Pod1 expression in tilapia persisted in differentiated juvenile and adult ovary and testis.Our data indicate for the first time that Pod1 is not only necessary for the onset of sexual differentiation,but also plays an important role in gonadal development in the teleost.

Genetic determination of osteoporosis in Chinese

Osteoporosis, one of the major and growing health problems around the world, is defined asa systemic SKeletal olsease cnaracterized by ,uw bone mass and microarchitectural deterioration of bone tissue, with high bone fragility and susceptibility to fractures. In China, 6.97% of the population （ 13 billion ） suffered from osteoporosis in 2000. As far as Hongkong was concerned,

The unsulfated extracellular N-terminus of vGPCR reduces the tumorigenicity of hGRO-α in nude mice

The Kaposi's Sarcoma-associated Herpesvirus (KSHV)-encoded G-protein coupled receptor (vGPCR) is an oncoprotein that is implicated in KSHV-associated malignancies. We previously revealed vGPCR incorporates sulfate groups within its extracellular N-terminal tyrosine residues (Y26 and Y28) and that this tyrosine sulfation is crucial for its tumorigenicity in nude mice. hGRO-binds vGPCR in a sulfotyrosine-dependent manner and promotes its tumorigenicity through autocrine signaling. Interestingly, an unsulfated vGPCR mutant (yydd-vGPCR) attenuated the tumor growth triggered by hGRO-α . In this study, the extracellular N-terminus of vGPCR (wt-vGN) and an unsulfated vGPCR mutant (yydd-vGN) were individually secreted, expressed and purified. A radioactive labeling assay demonstrated that wt-vGN but not yydd-vGN incorporated [ 35 S]-sulfate. In nude mice, NIH3T3 cells expressing yydd-vGN but not wt-vGN could significantly inhibit the tumor growth triggered by hGRO-α . All our data support the conclusion that the unsulfated extracellular N-terminus of vGPCR reduces the tumorigenicity of hGRO-α .

Mechanism of action of two insect toxins huwentoxin-Ⅲ and hainantoxin-Ⅵ on voltage-gated sodium channels

Selenocosmia huwena and Selenocosmia hainana are two tarantula species found in southern China.Their venoms contain abundant peptide toxins.Two new neurotoxic peptides,huwentoxin-Ⅲ(HWTX-Ⅲ) and hainantoxin-VI(HNTX-VI),were obtained from the venom using ion-exchange chromatography and reverse-phase high performance liquid chromatography(RP-HPLC).The mechanism of action of HWTX-Ⅲ and HNTX-VI on insect neuronal voltage-gated sodium channels(VGSCs) was studied via whole-cell patch clamp techniques.In a fashion similar to δ-atracotoxins,HNTX-VI can induce a slowdown of current inactivation of the VGSC and reduction in the peak of Na+ current in cockroach dorsal unpaired median(DUM) neurons.Meanwhile,10 μmol/L HNTX-IV caused a positive shift of steady-state inactivation of sodium channel.HWTX-ⅡI inhibited VGSCs on DUM neurons(concentration of toxin at half-maximal inhibition(IC50)≈1.106 μmol/L) in a way much similar to tetrodotoxin(TTX).HWTX-Ⅲ had no effect on the kinetics of activation and inactivation.The shift in the steady-state inactivation curve was distinct from other depressant spider toxins.The diverse effect and the mechanism of action of the two insect toxins illustrate the diverse biological activities of spider toxins and provide a fresh theoretical foundation to design and develop novel insecticides.

Autotriploid origin of Carassius auratus as revealed by chromosomal locus analysis

In the Dongting water system, the Carassius auratus(Crucian carp) complex is characterized by the coexistence of diploid forms(2n=100, 2n CC) and polyploid forms. Chromosomal and karyotypic analyses have suggested that the polyploid C. auratus has a triploid(3n=150, 3n CC) and a tetraploid origin(4n=200), respectively. However, there is a lack of direct genetic evidence to support this conclusion. In this paper, analysis of the 5S r DNA chromosomal locus revealed that the 3n CC is of triploid origin. Analysis of the species-specific chromosomal centromere locus revealed that 3n CC individuals possess three sets of C. auratus-derived chromosomes. Our results provide direct cytogenetic evidence suggesting that individuals with 150 chromosomes are of autotriploid origin within the C. auratus complex. It marks an important contribution to the study of polyploidization and the evolution of vertebrates.

Multiple analyses indicate the specific association of NR1I3,C6 and TNN with low hip BMD risk

Low hip bone mineral density(BMD)is an important index for osteoporosis and is associated with hip fracture,which leads to more cases of disability and mortality than all other kinds of fractures(Kanis et al.,2007).BMD’s heritability is more than 60%(Arden et al.,1996).A number of candidate loci for BMD have been previously identified by Genome Wide Association Studies(GWAS)(Xiong et al.,2009;Karasik et al.,2010;Zhang et al.,2013).Nevertheless,many significant signals based on GWAS are