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Jun Zou

Publications and source records attributed to Jun Zou.

At least 37 records · Page 2Linked to original sources
PubMed2004

Data mining techniques for cancer detection using serum proteomic profiling.

OBJECTIVE: Pathological changes in an organ or tissue may be reflected in proteomic patterns in serum. It is possible that unique serum proteomic patterns could be used to discriminate cancer samples from non-cancer ones. Due to the complexity of proteomic profiling, a higher order analysis such as data mining is needed to uncover the differences in complex proteomic patterns. The objectives of this paper are (1) to briefly review the application of data mining techniques in proteomics for cancer detection/diagnosis; (2) to explore a novel analytic method with different feature selection methods; (3) to compare the results obtained on different datasets and that reported by Petricoin et al. in terms of detection performance and selected proteomic patterns. METHODS AND MATERIAL: Three serum SELDI MS data sets were used in this research to identify serum proteomic patterns that distinguish the serum of ovarian cancer cases from non-cancer controls. A support vector machine-based method is applied in this study, in which statistical testing and genetic algorithm-based methods are used for feature selection respectively. Leave-one-out cross validation with receiver operating characteristic (ROC) curve is used for evaluation and comparison of cancer detection performance. RESULTS AND CONCLUSIONS: The results showed that (1) data mining techniques can be successfully applied to ovarian cancer detection with a reasonably high performance; (2) the classification using features selected by the genetic algorithm consistently outperformed those selected by statistical testing in terms of accuracy and robustness; (3) the discriminatory features (proteomic patterns) can be very different from one selection method to another. In other words, the pattern selection and its classification efficiency are highly classifier dependent. Therefore, when using data mining techniques, the discrimination of cancer from normal does not depend solely upon the identity and origination of cancer-related proteins.

Biomarkers, Tumor↗
PubMed2004

Analysis and characterisation of IL-1beta processing in rainbow trout, Oncorhynchus mykiss.

Mammalian IL-1beta is produced as a biologically inactive 31 kDa precursor, which is converted to the active 18 kDa form by proteolytic processing. Synthesis and processing of native piscine IL-1beta is poorly understood. In the present study, the native IL-1beta precursor or mature peptides were detected at sizes of approx. 29 kDa and 24 kDa in cell lysates of a rainbow trout macrophage cell line RTS-11, with or without LPS stimulation, by Western blot analysis using a polyclonal antibody against the putative trout mature IL-1beta (rmIL-1beta) produced in Escherichia coli. Processing of the 29 kDa precursor into a 24 kDa mature peptide was confirmed by analysis of such proteins using a monoclonal conjugate (Ni-NTA-HRP) against 6 histidines in lysates of the RTS-11 cells transfected with an expression plasmid containing the IL-1beta precursor molecule tagged with 6 histidines at its C terminus. Only the recombinant mature 24 kDa) IL-1beta/HIS protein was purified from the culture supernatants of the transfected cells, indicating the molecule is cleaved to be secreted. These findings strongly suggest that the trout IL-1beta molecule is processed in trout macrophages in an analogous way to the situation with mammalian IL-1beta despite the lack of a clear ICE cut site.

Animals↗
PubMed2004

Protease domain of human ADAM33 produced by Drosophila S2 cells.

Human ADAM33 is a multiple-domain, type-I transmembrane zinc metalloprotease recently implicated in asthma susceptibility [Nature 418 (2002) 426]. To provide an active protease for functional studies, expression of a recombinant ADAM33 zymogen (pro-catalytic domains, pro-CAT) was attempted in several insect cells. The pro-CAT was cloned into baculovirus under the regulation of the polyhedron promoter and using either the honeybee mellitin or ADAM33 signal sequence. Sf9 or Hi5 cells infected with these recombinant viruses expressed the majority of the protein unprocessed and as inclusion bodies ( approximately 10 mg/L). On the other hand, similar constructs could be expressed, processed, and secreted by Drosophila S2 cells using a variety of constitutive (actin, pAc5.1) or inducible (metallothionein, PMT) promoters and leader sequences (e.g., native and BiP). Higher expression level of 10-fold was observed for the inducible system resulting in an average yield of 20 mg/L after purification. The majority of the catalytic domain purified from the Drosophila conditioned media remained associated with the pro-domain after several chromatography steps. An induction cocktail containing cadmium chloride and zinc chloride was subsequently developed for the PMT system as an alternative to using cupric sulfate or cadmium chloride as single inducers. The novel induction cocktail resulted in an increased ratio of secreted catalytic to pro-domain, and yielded milligram amounts of highly purified protease. The availability of this modified expression system facilitated purification of the wild type and several glycosylation mutants, one of which (N231Q) crystallized recently for X-ray structure determination [J. Mol. Biol. 335 (2003) 129].

ADAM Proteins↗
PubMed2004

Discovery and characterization of two types of liver-expressed antimicrobial peptide 2 (LEAP-2) genes in rainbow trout.

The sequences and gene organisation of two LEAP-2 molecules (LEAP-2A and LEAP-2B) from rainbow trout, Oncorhynchus mykiss are presented. Both genes consist of a 3 exon/2 intron structure, with exon sizes comparable to known mammalian genes. LEAP-2A notably differs from LEAP-2B in having larger introns and a larger 3' UTR. The predicted proteins contain a signal peptide and prodomain, followed by a mature peptide of 41 aa containing four conserved cysteines. The RXXR cleavage site to release the mature peptide was also conserved. Both genes were found to be constitutively expressed in the liver, with expression in the intestine, and to a lesser extent the skin, evident after bacterial challenge.

Aeromonas↗
PubMed2004

Sequencing and expression of the second allele of the interleukin-1beta1 gene in rainbow trout (Oncorhynchus mykiss): identification of a novel SINE in the third intron.

A lambda clone containing a rainbow trout IL-1beta1 gene was isolated by a PCR screening strategy from a genomic library cloned in lambda GEM-11, and an EcoRI fragment from this clone was fully sequenced, and contained 1680 bp 5'-flanking sequence, the whole IL-1beta1 gene open reading frame, and the 3'-flanking region with two potential poly A signals and poly A sites. This clone encoded a protein that shared 99.8% identity to the previously published trout IL-1beta1 cDNA sequence, with only three base substitutions. The main difference was that this clone had an additional complete HpaI SINE insertion in the 3rd intron making intron III 211 bp larger (834 bp via 623 bp). Thus this sequence was designated as allele B (Big intron III) of IL-1beta1 and the previously reported sequence as allele S (Short intron III). Three lines of evidence (allele specific PCR, cloning and sequencing, and direct sequencing of PCR products) revealed that allele B was constitutively expressed and could respond to stimulation with lipopolysaccharide or trout recombinant IL-1beta. Searching of the GenBank database with the HpaI SINE sequence resulted in three additional HpaI loci being identified in rainbow trout. Another SINE retroposition was also identified in the same intron of both alleles of IL-1beta1 by comparison with the trout IL-1beta2 gene. This novel SINE sequence, sharing high homology with the HpaI SINE at the 3'-end region, is present in EST databases of several species including human, mouse and fish. The consensus of this novel SINE shares 57 to 61% identities to tRNA-Leu from different species. Another older retroposition event in the same intron of IL-1beta1 has also been hypothesised, recognised as six adenines, that may function as a RNA polIII terminator. A model for the IL-1beta1 allele formation is proposed. Following the earliest retroposition into one of the two IL-1beta genes that resulted from a genome duplication in salmonids, the proper environment for successive PV SINE retroposition was created. A recent retroposition of the HpaI SINE in IL-1beta1 resulted in the formation of the two alleles of IL-1beta1. Examination of the SINEs insertion and their host gene microenvironments revealed that the SINE retroposition does not appear random, both in the site selection and the direction of insertion. The mechanism governing this outcome is discussed.

Alleles↗
PubMed2004

Effective combinatorial strategy to increase affinity of carbohydrate binding by peptides.

The Thomsen-Friedenreich antigen, a carcinoma-associated disaccharide involved in carcinoma cell homotypic aggregation and increased metastatic potential, has clinical value as a prognostic indicator and a marker of metastasized cells. Hence, it can reasonably be predicted that antigen-binding macromolecules are valuable clinical in vivo diagnostic/therapeutic targeting agents. Recently, we have selected first-generation antigen-binding peptides from a random peptide bacteriophage display library and have applied combinatorial affinity maturation to select functionally-maturated peptides, which target cultured carcinoma cells and inhibit carcinoma cell aggregation. In the current study we hypothesize that a targeted search of sequence space surrounding the antigen-binding consensus sequence will select unpredictable amino acid sequences in the non-consensus portions of the peptides, leading to increased affinity for the carbohydrate and greater solubility in physiological buffers. This comprehensive in vitro analysis demonstrates that preferential evolution of the amino-terminal sequence of the peptides occurred, which correlated, in structure/function studies, with the acquisition of maturated function. The maturated peptides are more soluble than the earlier peptides. Studies of peptide binding to the disaccharide indicate that two maturated peptides (P-30-1, F03) have higher affinity for the antigen and bind with higher intensity to the surface of cultured human carcinoma cells than the first-generation peptides. The results support our hypothesis that affinity maturation can improve carbohydrate binding by peptides and have theoretical importance as the first report of maturation of carbohydrate-binding affinity in a small, soluble peptide.

Amino Acid Sequence↗
PubMed2004

Biodistribution of filamentous phage peptide libraries in mice.

In vivo phage display is a new approach to acquire peptide molecules that bind stably to a given target. Phage peptide display libraries have been selected in mice and humans and numerous vasculature-targeting peptides have been reported. However, in vivo phage display has not typically produced molecules that extravasate to target specific organ or tumor antigens. Phage selections in animals have been performed for very short times without optimization for biodistribution or clearance rates to a particular organ. It is hypothesized that peptides that home to a desired antigen/organ can be obtained from in vivo phage experiments by optimization of incubation times, phage extraction and propagation procedures. To accomplish this goal, one must first gain a better understanding of the in vivo biodistribution and rate of clearance of engineered phage peptide display libraries. While the fate of wild type phage in rodents has been reported, the in vivo biodistribution of the commonly used engineered fd-tet M13 phage peptide display libraries (such as in the fUSE5 vector system) have not been well established. Here we report the biodistribution and clearance properties of fd-tet fifteen amino acid random peptide display libraries in fUSE5 phage in three common mouse models employed for drug discovery - CF-1, nude, and SCID mice.

Animals↗
PubMed2004

Identification and expression analysis of an IL-18 homologue and its alternatively spliced form in rainbow trout (Oncorhynchus mykiss).

A homologue of interleukin 18 has been identified from rainbow trout, Oncorhynchus mykiss. The trout IL-18 gene spans 3.7 kb and consists of six exons and five introns, sharing the same gene organization with its human counterpart. The putative translated protein is 199 amino acids in length with no predicted signal peptide. Analysis of the multiple sequence alignment reveals a conserved ICE cut site, resulting in a mature peptide of 162 amino acids. The trout IL-18 shares 41-45% similarity with known IL-18 molecules and contains an IL-1 family signature motif. It is constitutively expressed in a wide range of tissues including brain, gill, gut, heart, kidney, liver, muscle, skin and spleen. Transcription is not modulated by lipopolysaccharide, poly(I:C) or trout recombinant IL-1beta in primary head kidney leucocyte cultures and RTS-11 cells, a macrophage cell line. However, expression is downregulated by lipopolysaccharide and rIL-1beta in RTG-2 cells, a fibroblast-like cell line. An alternatively spliced form of IL-18 mRNA has also been found and translates into a 182 amino acid protein with a 17 amino acid deletion in the precursor region of the authentic form. This alternatively spliced form is also widely expressed although much lower than the authentic form. Interestingly, its expression is upregulated by lipopolysaccharide and poly(I:C), but is not affected by rIL-1beta in RTG-2 cells. The present study suggests that alternative splicing may play an important role in regulating IL-18 activities in rainbow trout.

Alternative Splicing↗
PubMed2004

Is phage display technology on target for developing peptide-based cancer drugs?

New tumor targeting agents are required to advance cancer diagnosis and treatment. Bacteriophage (phage) display technology, a molecular genetic means of combinatorial drug discovery, is an emerging approach to identify and improve peptide molecules as pharmaceuticals. Peptides are thought to have clinically desirable benefits over currently used biomolecules, such as antibodies, because of their rapid blood clearance, increased diffusion and tissue penetration, non-immunogenic nature and ease of synthesis. Using phage display, one can rapidly and simultaneously survey billion-clone peptide libraries, resulting in large numbers of "hits". However, only a few lead compounds resulting from the hits historically reach the drug market. Hence determining which peptide may best translate into a useful drug is of particular importance. Examination of successfully marketed drugs has highlighted key features of a winning agent, including low molecular weight, high affinity, stability, solubility, lipophilicity and conformational rigidity. Although peptide modulators of tumor cell function and cancer targeting agents have been developed, the majority of peptide-based drugs reported thus far are immune and cardiac regulators. In this review, we will highlight how phage display has been employed to isolate peptides that target key steps in cancer progression--from tumor growth to metastasis--and how phage display technology can be harnessed to select a priori peptides with inherent features essential for anti-cancer drug efficacy. In 2003, phage display provided us with several novel peptides not only in clinical trials but approved by the FDA for use as therapeutics in a variety of diseases--suggesting that the future looks bright for phage display in anti-cancer drug development.

Animals↗
PubMed2003

Catalytic activity of human ADAM33.

ADAM33 (a disintegrin and metalloproteinase) is an asthma susceptibility gene recently identified through a genetic study of asthmatic families (van Eerdewegh et al. (2002) Nature 418, 426-430). In order to characterize the catalytic properties of ADAM33, the metalloproteinase domain of human ADAM33 was expressed in Drosophila S2 cells and purified. The N-terminal sequence of the purified metalloproteinase was exclusively (204)EARR, indicating utilization of one of three furin recognition sites. Of many synthetic peptides tested as potential substrates, four peptides derived from beta-amyloid precursor protein (APP), Kit-ligand-1 (KL-1), tumor necrosis factor-related activation-induced cytokine, and insulin B chain were cleaved by ADAM33; mutation at the catalytic site, E346A, inactivated catalytic activity. Cleavage of APP occurred at His(14)/Gln(15), not at the alpha-secretase site and was inefficient (k(cat)/K(m) (1.6 +/- 0.3) x 10(2) m(-1) s(-1)). Cleavage of a juxtamembrane KL-1 peptide occurred at a site used physiologically with a similar efficiency. Mutagenesis of KL-1 peptide substrate indicated that the P3, P2, P1, and P3' residues were critical for activity. In a transfected cell-based sheddase assay, ADAM33 functioned as a negative regulator of APP shedding and mediated some constitutive shedding of KL-1, which was not regulated by phorbol 12-myristate 13-acetate activation. ADAM33 activity was sensitive to several hydroxamate inhibitors (IK682, K(i) = 23 +/- 7 nm) and to tissue inhibitors of metalloproteinase (TIMPs). Activity was inhibited moderately by TIMP-3 and TIMP-4 and weakly inhibited by TIMP-2 but not by TIMP-1, a profile distinct from other ADAMs. The identification of ADAM33 peptide substrates, cellular activity, and a distinct inhibitor profile provide the basis for further functional studies of ADAM33.

ADAM Proteins↗
PubMed2003

Expression in Escherchia coli and purification of sea bass (Dicentrarchus labrax) interleukin 1beta, a possible immunoadjuvant in aquaculture.

Interleukin 1beta (IL-1beta) is a pleiotropic cytokine that plays a pivotal role in regulating immune responses. Our group has recently cloned IL-1beta from sea bass (Dicentrarchus labrax), one of the main Mediterranean aquacultured fish species. The cDNA is 1292 bp and codes for a deduced peptide of 29.4 kDa with a pI of 5.1. As for trout and carp IL-1beta precursor sequence, no candidate cut site for ICE (IL-1beta converting enzyme) enzyme was apparent in the alignments of sea bass IL-1beta with other mammalian IL-1betas. Nevertheless, a possible mature peptide could start at Ala86, giving a protein of 176 amino acids. The nucleotide sequence coding for this polypeptide was cloned into a pQE-30 expression vector. The plasmid was then transformed in Escherichia coli, and the recombinant protein was purified. Finally, we demonstrated that this purified recombinant IL-1beta was able to induce IL-1beta gene expression in a dose-dependent manner on cells purified from sea bass head kidney and could have immunoadjuvant effects in sea bass vaccination experiments.

Animals↗
PubMed2003

[Laboratory and clinical study of levofloxacin against Helicobacter pylori].

OBJECTIVE: To assess antibacterial activity of levofloxacin to Helicobacter pylori (Hp) strains In Vitro and In Vivo. METHODS: The Minimum inhibitory concentration (MIC) for 52 clinical isolates was detected by agar dilution method and was compared with those of amoxicillin and clarithromycin. To examine the effects of pH variation on the susceptibility of Hp to levofloxacin, Mueller-Hinton agar with 7% defibrinated sheep blood was adjusted to a pH range of 4.0, 5.0, 7.0 by adding hydrochloric acid. 85 Hp-positive Patients with chronic active gastritis or active peptic ulcer disease were consecutively recruited in a prospective, open-label study. The enrolled patients were randomised to receive a seven-day course of omeprazole 20 mg bid plus amoxicillin 1000mg bid and levofloxacin 200mg bid. Their Hp status was assessed by (13)C-urea breath test and/or endoscopy 4 - 6 weeks after the end of treatment. RESULTS: The resistant rates of strains to levofloxacin, amoxicillin and clarithromycin were 1.9%, 11.5% and 25%, respectively. A dual-resistance to amoxicillin and clarithromycin was demonstrated in five Hp strains (9.6%), which were all susceptible to levofloxacin. The prevalence of strains with resistance to levofloxacin was lower than that of strains with resistance to clarithromycin (P < 0.01), and was no statistically different with amoxicillin (P > 0.05). The activity of levofloxacin was diminished under acidic environment (P < 0.01). 84 enrolled patients completed the study. 76 patients (PP and ITT analysis, 91.7%; 90.6%) become Hp-negative. Slight side-effects occurred in 5 patients (5.9%). CONCLUSION: In the present study, we report a high rate of resistance to amoxicillin and clarithromycin in this region. Omeprazole/levofloxacin-based triple therapy, including amoxicillin, is attractive because they combine a high eradication efficacy with an excellent tolerability and safety profile.

Adolescent↗
PubMed2003

Characterisation, expression and promoter analysis of an interleukin 10 homologue in the puffer fish, Fugu rubripes.

Using computer-based tools, an interleukin (IL) 10 homologue has been identified from the puffer fish ( Fugu rubripes) genome database. This is the first report on the existence of an IL-10 homologue in a non-mammalian vertebrate species. The Fugu IL-10 gene is located within a 2790-bp fragment including 549 bp of coding sequence which translates into an 183-amino-acid protein. It is predicted to contain five exons and four introns, sharing the same organization with the mammalian IL-10 genes. The size of the introns in the Fugu IL-10 gene is much smaller compared to mammalian IL-10 genes, whilst the size of the exons is similar. The deduced protein sequence shares 44-50% homology with the mammalian IL-10 sequences, 39-42% with the viral IL-10 sequences and 37-42% with other members of the IL-10 family, IL-20 and IL-22. Southern blot analysis indicates that a single copy of the IL-10 gene is present in the Fugu genome. A very low level of constitutive expression was detected in tissues of healthy fish including liver, kidney, gut and spinal cord, whilst no expression was detectable in spleen, gill, brain, gonad and eye. Analysis of the transcription regulation elements in the promoter region revealed that trans elements are located in the region between 1 bp and 721 bp, cis elements between 934 bp and 1114 bp and a tumour necrosis factor alpha responsive transcription element was located 92 bp upstream of the TATA box.

Amino Acid Sequence↗
PubMed2003

Human ADAM33 messenger RNA expression profile and post-transcriptional regulation.

We examined transcript expression and post-transcriptional regulation of human ADAM33, a recently identified asthma gene. A detailed messenger RNA (mRNA) expression profile was obtained using Northern, reverse transcription polymerase chain reaction, and in situ hybridization analyses. ADAM33 mRNA was expressed significantly in smooth muscle-containing organs, minimally in immune organs and hematopoietic cells, and highly in repairing duodenal granulation tissue. Expression was seen in asthmatic subepithelial fibroblasts and smooth muscle but not in respiratory epithelium. In all tissues, transcripts of approximately 5 kb predominated over those of approximately 3.5 kb by 2- to 5-fold. The effect of the 3' untranslated region (UTR) on ADAM33 protein expression and maturation was examined. The presence of the 3'UTR in untagged full-length constructs promoted prodomain removal, detected as mature approximately 100 kD protein by ADAM33-reactive antibodies; in its absence, maturation was 2- to 3-fold less in HEK293 cells. His-tagged and untagged constructs lacking the 3'UTR demonstrated that lack of maturation was not a result of tag-mediated effects. Minimal maturation of ADAM33 occurred in primary lung and MRC5 fibroblasts following adenoviral-mediated expression of ADAM33 lacking the 3'UTR. In contrast, prodomain removal was observed with plasmids and adenovirus encoding only the pro- and catalytic domains. Thus, the 3'UTR of ADAM33 and domains downstream of the catalytic domain regulate potential ADAM33 activity. Mechanisms of regulation of ADAM33, distinct from closely related ADAMs, thus include mRNA localization and processing and protein maturation.

3' Untranslated Regions↗
PubMed2003

Human ADAM33: protein maturation and localization.

ADAM33 (a disintegrin and metalloprotease) was recently found to be a novel asthma susceptibility gene. Domain-specific antibodies were used to study its expression and processing. When the pro-domain and catalytic domain were expressed by a stable-transfected cell line, the pro-domain was removed by cleavage within a putative furin cleavage site. The catalytic domain was active in an alpha(2)-macroglobulin complex formation assay and mutation of the catalytic site glutamic acid (E346A) eliminated activity. In transient transfections using the full-length protein, a pro-form and mature form were detectable and alternate glycosylation was demonstrated at sites within the catalytic domain. ADAM33 was detected on the cell surface, with the majority of protein detected intracellularly. The E346A mutation had no significant effect on protein processing. Endogenous ADAM33 was detected in bronchus tissue, bronchial smooth muscle cells, and MRC-5 fibroblasts, consistent with a role in the pathophysiology of asthma.

ADAM Proteins↗
PubMed2003

The effect of intraperitoneally administered recombinant IL-1beta on immune parameters and resistance to Aeromonas salmonicida in the rainbow trout (Oncorhynchus mykiss).

The present work provides the first information concerning the immunostimulatory activity of trout recombinant interleukin-1 beta (rIL-1beta) in vivo. The predicted rainbow trout mature IL-1beta peptide was produced as a recombinant protein in Escherichia coli. Optimal migration of peritoneal leucocytes and rIL-1beta induced phagocytosis occurred following intraperitoneal injection of 1 microg of the recombinant protein. Moreover, systemic IL-1beta, COX-2 and lysozyme II gene expression was enhanced following >or=1 microg rIL-1beta administration. Finally, resistance to Aeromonas salmonicida, the causative agent of furunculosis, was augmented at early times (2 days) post-injection of 1 microg rIL-1beta.

Adjuvants, Immunologic↗
PubMed2002

Cloning and functional characterisation of the interleukin-1 beta 1 promoter of rainbow trout (Oncorhynchus mykiss).

The upstream flanking region of the rainbow trout (Oncorhynchus mykiss) IL-1 beta 1 gene has been cloned and characterised functionally using luciferase-based reporter gene constructs, and the transcription start site (TSS) confirmed by RLM-RACE. A TATA box was present 27 bp upstream of the TSS, with an NF-kB site 19 bp upstream of the TATA box. Within 1217 bp of upstream sequence, 3 sites for NF-kB, 10 sites for NF-IL6, 15 sites for AP1, 6 sites for AP4, 2 sites for CHOP/CEBP alpha and 1 site for SP1 and PU.1 were identified. Seven potential sites for the transcription repressor Gfi-1 were also identified. Analysis of eight IL-1 beta 1s promoter luciferase constructs transfected into a trout fibroblast (RTG-2) cell line known to constitutively express IL-1 beta revealed that in the absence of intron 1, very low luciferase activity was detectable. All of the constructs containing intron 1 gave clear luciferase activity, with the highest luciferase activity detected with construct P2-4 containing 617 bp of upstream sequence. As little as 82 bp of upstream sequence gave relatively strong luciferase activity, a region containing both a PU.1 and NF-kB site. That NF-kB is a transcription factor required for expression of the trout IL-1 beta 1 gene was confirmed using inhibitor studies with lipopolysaccharide (LPS)-stimulated macrophages. Both trout recombinant IL-1 beta and LPS were able to increase luciferase activity in the reporter constructs, especially in those containing the most upstream sequence with the lowest constitutive expression. The possibility that an upstream repressor is functioning to inhibit constitutive expression of IL-1 beta in this species is discussed.

5' Flanking Region↗