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J L Ding

Publications and source records attributed to J L Ding.

At least 37 records · Page 2Linked to original sources
PubMed1998

A novel antitumour compound from the mucus of a coral, Galaxea fascicularis, inhibits topoisomerase I and II.

S2 isolated from the mucus of G. fascicularis contains anti-topoisomerase activity, causing cytotoxicity to a multiple-drug resistant leukaemia cell line, P388/VCR. It inhibits DNA relaxation catalysed by topoisomerase I and II. S2 stabilised the topoisomerase I-DNA cleavable complex, thereby inhibiting the DNA replication machinery, leading to cell death. The active centre of S2 probably contains a Gal beta(1-4)GlcNAc dissacharide linkage. S2 has potentials of being developed into a new anticancer therapeutic.

Animals↗
PubMed1996

Transcription regulatory signals in the 5' and 3' regions of Oreochromis aureus ER gene.

The Oreochromis aureus estrogen receptor (OaER) 5' region is 62.2% AT-rich and does not display well-positioned consensus TATA and CAAT boxes. A functional imperfect 13 bp ERE, TGTTAtggTGACC, deviating at 2 bases is located in the leader exon. Transient transfection assays indicate that this ERE confers a 5-fold increase in SEAP reporter gene activity at 20 h post E2-induction. The continued elevation of transcription after the initial peak could be responsible for the "memory' effect of E2-induced vitellogenesis. The 4.4 kb OaER 3' region is AT-rich and has a high representation of 1/2 EREs and GREs. There are 10 copies of the destabilizing pentamer ATTTA which, in transient transfection experiments strongly suppressed SEAP activity. The 3' EREs are functional, and on their own, induce a bimodal increase in SEAP activity of 9-fold at 6 h and 11-fold at 18 h post-E2 induction. A recombinant construct of SEAP gene flanked by the 5' upstream and 3' flanking regions of the OaER gene allowed these regulatory signals to "cross-talk' to achieve a 5- and 25-fold increase for the first and second peaks, respectively. This bimodal response to E2-induction is attributed to both transcriptional and translational controls over the SEAP gene. This work represents a novel illustration of a synergistic interaction between the 5' and 3' regulatory elements of a steroid receptor in autoregulation.

Animals↗
PubMed1996

The first contiguous estrogen receptor gene from a fish, Oreochromis aureus: evidence for multiple transcripts.

The O. aureus estrogen receptor (OaER) gene of 40.4 kb containing ten exons is the first complete piscine gene to be cloned. There are two extra introns: intron I that divides the 5' UTR into two exons, and intron V that intersperses D and E1 exons. Except for I and V, other introns have identical positions to those of human ER gene. All the donor and acceptor splice sites exhibit consensus sequences. The promoter lacks consensus TATA and CAAT boxes. This region exhibits several putative regulatory elements. A functional imperfect ERE deviating at two bases is located in the leader exon, thus suggesting that this gene is autoregulated. The OaER gene lacks an A region whereas its C and E domains are highly conserved. Within the ER subfamily, OaER exhibits the longest F domain of 77 amino acids. OaER has a long 3'UTR constituting >1/2 of its transcript. Using RT-PCR and SI nuclease mapping, we report for the first time the usage of both alternative transcriptional start sites and polyadenylation signals during estrogen-induced OaER expression. Thus, O. aureus may have four species of ER transcripts differing structurally in their transcriptional start sites and lengths of their 3' UTR.

Amino Acid Sequence↗
PubMed1996

Neuroblastoma cell culture assay shows that Carcinoscorpius rotundicauda haemolymph neutralizes tetrodotoxin.

Veratridine facilitates the influx of Na+ into Neuro-2A cells and this process is exacerbated by the presence of a Na+/K(+)-ATPase inhibitor, ouabain, leading to a reduction in cell viability. However, tetrodotoxin neutralizes the cytotoxic effects of veratridine, hence sustaining the viability of Neuro-2A cells. This neutralizing ability was negated when TTX was first reacted with cell-free haemolymph of Carcinoscorpius rotundicauda, resulting in reduced cell viability. These results therefore indicate a bona fide effect of the cell-free haemolymph against tetrodotoxin as demonstrated by in vitro cell culture technique.

Animals↗
PubMed1995

Molecular cloning and sequencing of the hormone-binding domain of Oreochromis aureus estrogen receptor gene.

The nucleotide sequence of the estrogen receptor gene of Oreochromis aureus (OaER) indicates that the hormone-binding E domain is composed of 4 exons interspersed by short introns of only 0.18-1.3 kb each. All 4 E exons exhibit consensus sequences flanking the donor and acceptor splice sites. Analysis of introns revealed (i) numerous palindromic and half-palindromic steroid responsive elements including ERE, TRE and GRE, (ii) six alternative polyadenylation signals and (iii) putative control regions identified by the clustering of transcription factor binding sites. Of particular interest is the presence of a TATA and CAAT box in intron IV. The hydropathicity profile shows that the E exons are relatively hydrophobic. Two receptor dimerization regions have been observed: a conserved heptad repeat of hydrophobic residues (R168-M193) and a perfect leucine zipper (L36-L57). The presence of multiple sites for kinase activity in these regions suggests the importance of phosphorylation in the regulation of receptor functions and ligand-affinity.

Amino Acid Sequence↗
PubMed1995

The hormone-binding domain of Oreochromis aureus estrogen receptor gene: homology comparison with other steroid binding receptors.

Although the estrogen receptor gene of Oreochromis aureus (OaER) shows 85% homology to rainbow trout ER (rtER), the molecular organization of its exons and introns in the hormone-binding E domain is more closely related to the human ER gene. Comparison with other vertebrates yielded reduced homologies of 64-67%, probably due to evolutionary speciation. The E1 and E2 exons of OaER are interspersed by a short intron of 1.3 kb which is flanked by consensus splice sites. This is in sharp contrast to the 11 kb intron separating E1 and E2 exons of rtER which also displayed a rare GC donor junction. Three conserved cys at 83, 112 and 195, which are important for formation of 3-D ligand-binding pocket were found in OaER. However, the 4th conserved cys is replaced by a ser. This substitution which is the result of a single base mutation probably suggests different affinity for estrogen or transactivation of the OaER gene. Two overlapping steroid binding and receptor dimerization domains have been observed. The E domain of OaER and rtER has diversified significantly from that of other non-piscine vertebrates, such that they form a separate subgroup in the UPGMA tree of steroid receptors.

Amino Acid Sequence↗
PubMed1995

Molecular cloning and sequence analysis of factor C cDNA from the Singapore horseshoe crab, Carcinoscorpius rotundicauda.

Two forms of Factor C cDNAs: CrFC21 (3448 bp) and CrFC26 (4182 bp) have been cloned into lambda gt22. CrFC26 includes 568 nucleotides of 5' untranslated region (5' UTR) containing seven ATGs before the real initiation site, an open reading frame (ORF) of 3249 nucleotides, a stop codon, and 365 nucleotides of 3' untranslated sequence. There are four polyadenylation signals and six potential glycosylation sites. The ORF codes for a signal peptide of 24 amino acids and a Factor C zymogen of 1059 residues. The CrFC21 lacks most of the 5' UTR, and has some base changes in its ORF. The predicted secondary mRNA structures of the 5' end of CrFC26 showed numerous stem-and-loop structures, thus obscuring its real start codon. In contrast, CrFC21 has a well-exposed AUG start site, and expresses Factor C in transcription-translation reactions in vitro. There is a typical serine protease catalytic triad of Asp-His-Ser, which is structurally like prothrombin, but catalytically more similar to trypsin. Although an overall homology of 97.7% was observed in comparison with the Tachypleus tridentatus Factor C (TtFC) cDNA, there were notable differences in the restriction sites and subtle base substitutions in the CrFC cDNA. The high degree of homology between Factor C from T. tridentatus and C. rotundicauda substantiates, at the molecular level, the proximity of these two species in the course of evolution. This finding contravenes the apparent disparities with respect to their morphology, ecological habitat, and taxonomical classification.

Amino Acid Sequence↗
PubMed1995

Expression of Carcinoscorpius rotundicauda factor C cDNA.

The cDNA encoding Factor C (FC) from the Singaporean horseshoe crab Carcinoscorpius rotundicauda has been studied for in vitro coupled transcription-translation (TnT) under the T7 promoter. Two species of full length cDNA, CrFC26 and CrFC21 which differ in length and nucleotide sequence at their 5' untranslated regions (UTR) were used in this study. Wild type CrFC26 with a long 5' UTR containing multiple "false" ATGs failed to generate a translated product. With a more accessible ATG codon in CrFC21, the recombinant construct gave a high yield of FC when transcribed and translated in vitro. CrFC26 deletion mutants which lack the entire 5' UTR and portions of the putative leader peptide were translatable, albeit at lower efficiency as compared to CrFC21. In vitro and in vivo expression of truncated portions of the CrFC21-T7 gene 10 fusions have been compared. In vitro reactions yielded single gene products from each of the expression constructs whereas E. coli produced three major immunoreactive bands of FC.

Animals↗
PubMed1994

Cortisol-induced hepatic vitellogenin mRNA in Oreochromis aureus (Steindachner).

Cortisol triggers rapid but transient transcription of the silent vitellogenin (Vg) gene in male Oreochromis aureus, producing short-lived Vg mRNA. Implantation of cocoa butter containing cortisol at 0.8, 8, 20, and 40 micrograms/g fish body wt induced hepatic Vg mRNA within 1 or 2 hr. Implantation of cortisol at the lowest of these dosages (0.8 micrograms/g body wt) produced a transient surge in serum cortisol within 1 hr. This is equivalent to a 30-fold increase over that of the resting male fish in which the endogenous cortisol levels measured 10.5 +/- 2.39 and 11.4 +/- 3.1 ng/ml, respectively, for fish kept communally or singly. Although sham implantation with cocoa butter also resulted in the elevation of serum cortisol, the rise was much more gradual, only reaching a peak after 6 hr. Thus, parameters like the treatment regime, sampling time intervals after implantation, and the interaction between these two factors can significantly affect the serum cortisol concentrations (P = 0.001). Although implantation of cocoa butter with or without cortisol increased serum cortisol, albeit unsynchronized, Vg mRNA was detected only in the hormone-treated group. The profile of Vg mRNA accumulation appeared to coincide with that of serum cortisol, further suggesting the activating effect of exogenous cortisol on Vg gene. Compared with an earlier study on estradiol-induced Vg gene expression, the action of cortisol was more rapid, but transient. This is reflected in the rapid clearance of the cortisol-induced Vg mRNA and, hence, the lack of plasma Vg protein during cortisol treatment.

Animals↗
PubMed1994

A tetrodotoxin neutralizing system in the haemolymph of the horseshoe crab, Carcinoscorpius rotundicauda.

The horseshoe crab, Carcinoscorpius rotundicauda, when injected intracardially with tetrodotoxin (TTX) at a dosage of 10 x LD50 of mice showed no mortality. The resulting haemolymph extracted 24 hr later from these crabs caused no fatalities when injected into mice. TLC analysis revealed that cell free haemolymph (CFH) had converted a single yellow fluorescent spot of TTX into two blue fluorescent components when the reaction was carried out at 25 degrees C. However, no fluorescence was observed when TTX was reacted with CFH at 37 degrees C. There was also an increase in the intensity of protein bands on SDS-PAGE gel corresponding to 26,000 and 28,000 mol. wts. This may be the result of the TTX-neutralization reaction in CFH. The pretreatment of TTX with CFH reduced the toxicity of TTX in mice by two-fold. The anti-TTX activity is temperature and time dependent as well as heat stable. Protein denaturation of the haemolymph suggests that proteinaceous factor(s) may also play a role in TTX-neutralizing activity of CFH. It is therefore possible that the detoxification system is attributed to concerted actions of both proteinaceous and non-proteinaceous factors in the haemolymph.

Animals↗
PubMed1994

Two major groups of vitellogenin cDNA clones from Oreochromis aureus (Steindachner).

cDNA libraries were constructed using livers of male and female Oreochromis aureus that were stimulated with estradiol. Restriction map analyses of vitellogenin (Vg) cDNA clones indicated 4 subgroups with related restriction patterns, represented by pOAVg2 (from female) and pOAVg50, 71 & 87 (from male) fish. With the exception of pOAVg2, all other clones obtained from female fish resembled pOAVg50. There are no internal repeats within these cDNA clones. Southern blot cross-hybridisations distinguished pOAVg87 (group A) from pOAVg2, 50 and 71 (group B). The existence of these 2 major groups of Vg cDNAs was further confirmed by dot blot hybridisations and hybrid melting assays under varying stringencies. The difference of melting temperature, Tm between the 2 groups suggests a 7% intraspecific divergence in the Vg gene family.

Animals↗
PubMed1993

Two forms of factor C from the amoebocytes of Carcinoscorpius rotundicauda: purification and characterisation.

The two apparent form of the endotoxin-sensitive Factor C which were found to exist in the amoebocytes of horseshoe crabs have been separately purified to homogeneity from the lysate of the South-East Asian species, Carcinoscorpius rotundicauda. Both forms are serine proteinase zymogens having an apparent molecular mass of 132 kDa. By reducing SDS-PAGE, one was shown to consist of a single polypeptide while the other has a heavy chain (80 kDa) and a light chain (52 kDa) bridged by disulfide linkage(s). Both zymogen forms have endotoxin (lipopolysaccharide) receptors to which endotoxin binds to activate their catalytic sites. However, single-chain Factor C appears to have higher-affinity endotoxin-binding sites which are competitively but reversibly occupied by DMSO when the latter was added during its purification. Another salient difference between the two forms of Factor C is exhibited in their manner of activation by endotoxin. While double-chain Factor C appears similar to that of Tachypleus tridentatus, single-chain Factor C did not undergo any proteolytic cleavage upon activation. This conformational transition of zymogen activation suggests an alternative reversible pathway of endotoxin activation for the single-chain Factor C.

Animals↗
PubMed1993

Morphological changes of Carcinoscorpius rotundicauda amoebocyte and Escherichia coli during their interaction.

Interaction between Carcinoscorpius rotundicauda amoebocytes and Escherichia coli or Staphylococcus aureus has shown that whilst E. coli was observed to lyse, S. aureus remained intact. The heavily granulated amoebocytes were activated by the lipopolysaccharide of E. coli to undergo dramatic morphological changes with time, leading to degranulation which caused lysis of the E. coli. Degranulation of the amoebocytes involved maturation of the larger electron dense granules (0.7-2.0 micron) which were apparently mobilised towards the cell membrane whilst undergoing a loss in electron density. Some of these larger granules were expelled at 15-30 min while other intracellular granules underwent further degranulation, where parallel filaments appearing like microtubules were evident, giving the granules a striated and less dense form. By 60 min, there was complete depolymerisation of the microtubular filaments giving rise to undefined, fused and homogeneously packed striations and particulate material in the cytoplasm; the smaller electron dense granules (0.3-0.7 micron) remained unchanged. This asynchronous degranulation and preferential expulsion of granules from only some amoebocytes while maintaining others intact, implies that the horseshoe crab has safeguards in its defence mechanism, hence ensuring its continued survival as a 'living fossil'.

Animals↗
PubMed1993

Electrophoretic analysis of endotoxin-activated gelation reaction of Carcinoscorpius rotundicauda amoebocyte lysate.

Electrophoretic analysis of a 60 min reaction between E. coli endotoxin and the amoebocyte lysate showed that the coagulation reaction was complete by 15 min, with the conversion of coagulogen (21 kDa) to coagulin (17 kDa). Coincident with this observation was the maximal activities at 15 min, of Factor C and proclotting enzyme. On agitation of the coagulin gel clots, bioactive endotoxin was recovered. Densitometric scan of the electrophoretically-resolved proteins showed that the sum of coagulogen and coagulin remained almost constant at various time intervals of the coagulation reaction. Electrophoresis serves as a convincing and visually discernible method of studying the kinetics of coagulation, and defining the onset and completion of gelation. Furthermore, it is a useful means of examining the integrity of fresh lysate preparations based on the presence or absence of the 17 kDa coagulin band.

Animals↗
PubMed1991

Estradiol-induced vitellogenin gene expression in a teleost fish, Oreochromis aureus.

Vitellogenesis presents a versatile model for the study of hormone-induced gene expression. We report here the effects of estradiol-17 beta-propionate on vitellogenin gene expression in male Oreochromis aureus, a teleost fish. Vitellogenin mRNA of 6500 nucleotides has been elucidated from the livers of female and estradiol-treated male O. aureus. By hybridization with a specific O. aureus cDNA probe, the vitellogenin mRNA transcript was detected as early as 1 hr following primary and secondary estradiol-stimulations, although for the latter, the rate of accumulation of vitellogenin-specific mRNA was 20-fold higher. The vitellogenin mRNA peaked at 72 and 48 hr, respectively, for primary and secondary stimulations. At the translational level, the increase in plasma vitellogenin was further enhanced during the secondary stimulation. There was a distinct shift in the peak of plasma vitellogenin from Day 14 in the primary induction to Day 3 in the secondary stimulation. The plasma vitellogenin presented in two forms, 300 and 500 kDa, both of which were immunologically confirmed by Western blot analysis to be vitellogenin proteins.

Animals↗
PubMed1990

Inactivation of factor C by dimethyl sulfoxide inhibits coagulation of the Carcinoscorpius amoebocyte lysate.

The inhibition by dimethyl sulfoxide of the coagulation of the Carcinoscorpius Amoebocyte Lysate was found to be due to the inactivation of Factor C enzyme in the coagulation cascade and not due to the inactivation of proclotting enzyme as earlier reported in studies done on Limulus. Kinetic studies on both purified enzymes revealed that dimethyl sulfoxide completely but reversibly inhibited the activation of Factor C by endotoxins in a non-competitive manner whereas, it did not inhibit, albeit retard the activity of proclotting enzyme. This result also explains why clotting enzyme was shown to be largely unaffected by dimethyl sulfoxide.

Amino Acid Sequence↗
PubMed1990

Cloning, characterisation and expression of vitellogenin gene of Oreochromis aureus (Teleostei, Cichlidae).

cDNA synthesised from liver Poly A+ RNA of estradiol-stimulated male Oreochromis aureus was ligated to PTZ-19R and transformed into E. coli. TG-1 to yield 6 x 10(4) clones/ug DNA, of which 6% was vitellogenin positive. Restriction maps indicate 4 possible subgroups of vitellogenin cDNA, with homology, observed by Southern hybridisation, at a fragment flanked by Pst 1 sites. When the amino acid sequence derived from the DNA sequence of pOA Vg 62 was aligned with that of S. gairdneri, X. laevis, C. elegans and D. melanogaster, homologies of 63%, 22%, 21% and 15%, respectively, were obtained. Estradiol induced a vitellogenin gene transcript of 6500 nucleotides at 6 h and reached a maximum at 72 h after stimulation.

Amino Acid Sequence↗