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T J Lam

Publications and source records attributed to T J Lam.

At least 19 recordsLinked to original sources
PubMed2005

Molecular characterization of a major outer capsid protein encoded by the Threadfin aquareovirus (TFV) gene segment 10 (S10).

Genome segment 10 (S10) of Threadfin aquareovirus (TFV) was cloned, sequenced, analyzed and found to be 987 bp long encoding a protein of 298 aa with a predicted molecular mass of 32.0 kDa. The TFV S10 gene possesses terminal motifs, (5' GTTTTA and ATTCATC 3') which are also conserved in the S6 and S11 TFV gene segments. Sequence comparison revealed that the TFV S10 gene was similar to the Striped bass reovirus (SBR) VP7 outer capsid protein (OCP). A conserved putative zinc-finger motif, CCHC, present in the mammalian reovirus (MRV) delta3 protein, was identified in TFV and other aquareovirus VP7 protein. Phylogenetic analysis of the TFV VP7 protein indicated that TFV is closely related to SBR and Chum salmon reovirus (CSV) and possibly belong to the same species Aquareovirus A as SBR and CSV. The TFV VP7 protein was expressed in E. coli, purified and injected into mice. Serum specific antibodies were generated, however, the serum showed weak neutralizing activity. In contrast, co-incubation of this serum with another serum obtained from mice immunized with another OCP encoded by the TFV S6 gene segment resulted in a highly elevated antibody neutralization titer.

Amino Acid Sequence↗
PubMed2005

Effects of thyroid hormone on the development of immune system in zebrafish.

Effects of thyroxine (T4) and methimazole (MMI) on the development of the zebrafish immune system were investigated using continuous immersion treatment experiments. The effects of the treatments on thymus development were determined using computer-aided thymus morphometric analyses on in situ hybridization serial sections of the thymus while the effects on immune-related gene expression levels were monitored using quantitative real-time PCR. The findings indicate that thymus development and thymopoiesis, as indicated by thymus size, thymus Rag-1-positive region, and TCRAC expression level, were affected by T4 and MMI-treatments. With the exception of Ikaros, MMI-treated fish has lower immune-related gene expression levels, although it is not certain whether the effect resulted indirectly from the concomitant growth-retardation and/or directly from an effect on lymphopoiesis itself. The findings were comparable with those in mammalian system, thus providing the first evidence that the thyroid relationship with thymus development and lymphopoiesis is likely to be conserved from fish to higher vertebrates. It suggests the possibility of using zebrafish as a model system to investigate the molecular mechanisms involved in thyroid hormone-dependent disorders in the immune system.

Animals↗
PubMed2005

Single-cell protein diet of a novel recombinant vitellogenin yeast enhances growth and survival of first-feeding tilapia (Oreochromis mossambicus) larvae.

Yeast single-cell protein (SCP) is a high-nutrient feed substitute. This study evaluates the dual applications of a novel recombinant Pichia pastoris SMD1168H (SMD) yeast, expressing a tilapia vitellogenin protein (rVtg), as an SCP diet for Artemia and the first-feeding fish larvae. Instar II Artemia fed rVtg, rVtg precultured in 5% fish oil (rVtg-FO), Saccharomyces cerevisiae (SC), or native SMD had greater lipid contents (P < 0.05) than the freshly hatched. Lipid deposition in the Artemia fed rVtg or rVtg-FO was greater (P < 0.05) than in those fed SMD or SC. Diet-induced accumulation of low levels of docosahexaenoic acid [22:6(n-3)] was detected only in Artemia fed the rVtg-based diets. Tilapia (Oreochromis mossambicus) larvae were fed solely yeast diets singly or in combination (d 3-22), or a staggered regimen of yeast (d 3-12) followed by unenriched or yeast-enriched Artemia (d 13-22). The larvae fed rVtg for 22 d increased in length and weight (P < 0.05), whereas those fed SC or SMD suffered growth suppression and high mortality. Such adverse consequences were ameliorated when 50% of SC was substituted with rVtg. The larvae prefed rVtg followed by a dietary switch to Artemia preenriched for 48 h with rVtg or rVtg-FO were greatest in length, had the highest weight gain, and lived the longest. Besides delivering rVtg protein, essential fatty acids and amino acids, rVtg may have probiotic effects in enhancing larval survival. This study suggests the feasibility of using the rVtg yeast as an Artemia booster and an SCP first feed for larvae.

Animals↗
PubMed2004

Development of a rapid, sensitive and specific diagnostic assay for fish Aquareovirus based on RT-PCR.

A rapid, sensitive and highly specific detection method for Aquareovirus based on reverse-transcription polymerase chain reaction (RT-PCR) was developed. Based on multiple sequence alignment of the cloned sequences of a local isolates, the Threadfin reovirus (TFV) and Guppy reovirus (GPV) with Grass carp reovirus (GCRV), a pair of degenerate primers was selected carefully and synthesized. Using this primer combination, only one specific product, approximately 450 bp in length was obtained when RT-PCR was carried out using the genomic double-stranded RNA (dsRNA) of TFV, GPV and GCRV. Similar results were also obtained when Chum salmon reovirus (CSRV) and Striped bass reovirus (SBRV) dsRNA were used as templates. No products were observed when nucleic acids other than the dsRNA of the aquareoviruses described above were used as RT-PCR templates. This technique could detect not only TFV but also GPV and GCRV in low titer virus-infected cell cultured cells. Furthermore, this method has also been shown to be able to diagnose GPV-infected guppy (Poecilia reticulata) that exhibit clinical symptoms as well as GPV-carrier guppy. Collectively, these results showed that the RT-PCR amplification method using specific degenerate primers described below is very useful for rapid and accurate detection of a variety of aquareovirus strains isolated from different host species and origin.

Animals↗
PubMed2004

Characterization of the seabass pancreatic alpha-amylase gene and promoter.

Seabass (Lates calcarifer) pancreatic alpha-amylase gene was cloned and characterized. The alpha-amylase cDNA has 1620 bp and the deduced polypeptide has 522 amino acids. Southern blot indicated that there are two gene copies in the seabass genome. Sequence analysis showed that except for the loss of an intron in seabass, the coding region and the exon/intron boundaries are highly homologous to those of mammalian amylases. However, the promoter regions are distinctively divergent. To investigate the seabass amylase promoter, a series of deletion mutants was generated and fused to the luciferase reporter gene, followed by studies of their functional activity in rat AR42J cell line. Besides identifying several potential regulatory elements that have been previously identified in the human and mouse pancreatic amylase promoter, we have identified a glucocorticoid response element (GRE). However, while the human and mouse pancreatic amylase promoters are highly homologous between nucleotide -160 and transcription start site where GRE is located, the 5' promoter deletion mutants revealed that the GRE of the seabass amylase promoter was located far upstream -947 to -776 bp of the promoter. Site-directed mutagenesis of the putative GRE and electrophoretic mobility shift assays (EMSA) confirmed that this region was responsible for dexamethasone induction. However, no functional PTF-1 binding site, which is responsible for pancreas-specific transcription in higher vertebrates, was identified in seabass amylase promoter. Instead a Hepatocyte Nuclear Factor 3 binding site was found to modulate the amylase promoter expression. The evolutionary significance of this divergence in promoter regulation between seabass and mammals requires further studies.

Animals↗
PubMed2004

Expression of recombinant vitellogenin in the yeast Pichia pastoris.

Vitellogenin (Vtg) plays vital roles as precursor to the yolk proteins and as carrier for lipids, carbohydrates, phosphates, metal ions, vitamins, and hormones into the oocytes during the massive deposition of yolk nutrients for subsequent nourishment of the developing embryos. Reproductive success is highly sensitive to the nutritional quality of the broodstock diet, which greatly affects the egg and larval viability. We present a novel strategy for genetically engineering a Pichia pastoris yeast strain that constitutively produces recombinant Vtg (rVtg), for application as an enriched feed. The tilapia Oreochromis aureus Vtg (OaVtg) cDNA (5.3 kb) was cloned into a nonsecretory pGAPZA vector. Clones containing up to 31 copies of glyceraldehyde-3-phosphate dehydrogenase (GAP)-promoter-driven Vtg expression cassettes were isolated. These clones expressed a membrane-associated intracellular rVtg protein of 194 kDa, constituting up to 1.16% of total protein. To facilitate future purification of rVtg, we explored the possibility of secreting rVtg using the native Vtg secretion signal and the alpha-factor secretion signal of Saccharomyces cerevisiae. However, neither signal promoted the secretion of rVtg. The clones maximally expressed rVtg at 23 degrees C, reaching a peak at 22 h in shake flasks and 16 h in a fermentor. The clones exhibited a significant increase in essential amino acids and long-chain polyunsaturated fatty acids, which are important for its application as a high-quality nutrient feed.

Amino Acids, Essential↗
PubMed2004

Hormonal influence on amylase gene expression during Seabass (Lates calcarifer) larval development.

alpha-Amylase gene expression was detected in newly hatched seabass (Lates calcarifer) larvae and peaked at around first feeding. This suggests a greater importance of carbohydrates during early larval development than might be expected for carnivorous fishes. In vivo cortisol and triiodothyronine (T(3)) treatment of seabass larvae upregulated alpha-amylase gene expression. The identification of a functional glucocorticoid-response element (GRE) on the amylase gene promoter indicates that cortisol (glucocorticoid) stimulation of amylase gene expression is direct via GRE. However no TRE (thyroid-response element) was found on the amylase gene and its promoter, and various concentrations of T(3) (1nM-10microM) also did not induce alpha-amylase gene promoter activity in rat AR42-J cells transfected with the promoter construct, unlike dexamethasone treatment. This suggests that T(3) stimulation of amylase gene expression in vivo was indirect, probably secondary to its promotion of one or more developmental processes.

Amylases↗
PubMed2004

Development and maturation of the immune system in zebrafish, Danio rerio: a gene expression profiling, in situ hybridization and immunological study.

The development and maturation of the immune system in zebrafish was investigated using immune-related gene expression profiling by quantitative real-time polymerase chain reaction, in situ hybridization (ISH), immunoglobulin (Ig) detection by immuno-affinity purification and Western blotting as well as immersion immunization experiments. Ikaros expression was first detected at 1 day post-fertilization (dpf) and thereafter increased gradually to more than two-fold between 28 and 42dpf before decreasing to less than the initial 1dpf expression level in adult fish (aged 105dpf). Recombination activating gene-1 (Rag-1) expression levels increased rapidly (by 10-fold) between 3 and 17dpf, reaching a maximum between 21 and 28dpf before decreasing gradually. However, in adult fish aged 105dpf, the expression level of Rag-1 had dropped markedly, and was equivalent to the expression level at 3dpf. T-cell receptor alpha constant region and immunoglobulin light chain constant region (IgLC) isotype-1, 2 and 3 mRNAs were detected at low levels by 3dpf and their expression levels increased steadily to the adult range between 4 and 6 weeks post-fertilization (wpf). Using tissue-section ISH, Rag-1 expression was detected in head kidney by 2wpf while IgLC-1, 2 and 3 were detected in the head kidney and the thymus by 3wpf onwards. Secreted Ig was only detectable using immuno-affinity purification and Western blotting by 4wpf. Humoral response to T-independent antigen (formalin-killed Aeromonas hydrophila) and T-dependent antigen (human gamma globulin) was observed in zebrafish immunized at 4 and 6wpf, respectively, indicating that immunocompetence was achieved. The findings reveal that the zebrafish immune system is morphologically and functionally mature by 4-6wpf.

Animals↗
PubMed2003

Characterization of a novel ranavirus isolated from grouper Epinephelus tauvina.

A large icosahedral virus was isolated from diseased grouper Epinephelus tauvina. The virus grew well in several cultured fish cell lines, with stable and high infectivity after serial passages in grouper cell line (GP). The virus was sensitive to both acid and heat treatments. Virus replication was inhibited by 5-iodo-2-deoxyuridine (IUDR), indicative of a DNA-containing genome. The virus infectivity was reduced with ether treatment, suggesting that the virus was lipid-enveloped. Electron micrographs showed abundant cytoplasmic icosahedral virons in the virus-infected GP cells. The size of the intracellular nucleocapsid was 154 nm between the opposite sides, or 176 nm between the opposite vertices with an inner electron-dense core of 93 nm. Virus particles were released through budding from plasma membranes with a size of 200 nm in diameter. SDS-PAGE of purified virus revealed 20 structural protein bands and a major capsid protein (MCP) of 49 kDa. A DNA fragment of approximately 500 nucleotides was successfully amplified by polymerase chain reaction (PCR) using the primers from conserved regions of the MCP gene of frog virus 3 (FV3), the type species of Ranavirus. Subsequent multiple alignment and phylogenetic analysis showed that the newly isolated grouper virus was closely related to largemouth bass virus (LMBV), FV3 and Regina ranavirus (RRV). Our data suggests that the virus isolate is a novel member of genus Ranavirus, family Iridoviridae. We tentatively name the virus as Singapore grouper iridovirus (SGIV). SGIV was able to cause serious systemic disease capable of killing 96% of grouper fry.

Amino Acid Sequence↗
PubMed2003

Nodavirus infection in freshwater ornamental fish, guppy, Poicelia reticulata--comparative characterization and pathogenicity studies.

Biochemical, genomic and serological studies were carried out to characterize a virus obtained from diseased guppy, Poicelia reticulata. The SDS-PAGE analysis of CsCl purified virus showed two distinct bands with molecular weight of 42 kDa and 110 kDa. A 1367 nucleotide region of the coat protein gene was sequenced, which includes one full open reading frame of 1017 nucleotides and a region of 350 nucleotides at the 3'end. The nucleotide identity of this strain with the nodavirus isolated from Epinephelus tauvina (Singapore strain) is 98% and with other strains of fish nodaviruses the identity is more than 75%. Western blot analysis using rabbit antisera raised against the nodavirus from marine fish, E. tauvina confirmed its antigenic similarity to the marine nodavirus isolate. Asymptomatic infection in guppy fry was observed following experimental infection with this virus and the marine nodavirus isolate (Singapore strain) implying the spread of virus from marine fish to freshwater fish. This report forms the first description of a nodavirus infection in freshwater fish.

Amino Acid Sequence↗
PubMed2002

Morphologic transformation of the thymus in developing zebrafish.

The morphologic transformation of the developing zebrafish thymus from 1 week postfertilization (wpf) to 15 wpf is described. The thymus overall morphology changed from a small pouch-like shape at 1 wpf to a conical shape between 2 and 3 wpf before acquiring a more complex shape from 4 wpf onward. Rapid growth rate along the lateral axis at the region near the pharyngeal epithelium occurred between 1 and 2 wpf, whereas rapid growth rate along the dorsal-ventral axis occurred between 3 and 6 wpf. Expansion of thymocyte population beginning from 1 wpf became more evident by 2 to 3 wpf, as indicated by the apparent increase of different sizes of lymphocytes, recombination activating gene-1 (rag-1), and T-cell antigen receptor alpha chain constant region (TCRAC) -positive cells. Tissue section in situ hybridization (ISH) analysis with rag-1 probe reveals that cortex-medullary regionalization has begun between 1 and 2 wpf as rag-1 expression clearly demarcated the cortex, whereas the medulla was rag-1 negative. The presence of TCRAC-positive cells in the medulla by 2-3 wpf, suggests that the thymic selection processes had begun. The zebrafish thymus is morphologically mature by 3 wpf. Early signs of thymic involution were observed in zebrafish aged 15 wpf.

Animals↗
PubMed2002

Increased 21-hydroxylase and shutdown of C(17,20) lyase activities in testicular tissues of the grouper (Epinephelus coioides) during 17alpha-methyltestosterone-induced sex inversion.

The metabolism in vitro of [(3)H]17-hydroxyprogesterone by gonadal tissues of the grouper (Epinephelus coioides) during 17alpha-methyltestosterone (MT)-induced female-to-male sex inversion was examined. In the female phase, C(17,20) lyase, 5beta-reductase, 3alpha/beta-HSD, 20beta-HSD, and 17beta-HSD activities resulted in the biosynthesis of 5beta-pregnans and 5beta-androstanes (including 5beta-androstane-3alpha/beta, 17beta-diol, 3alpha/beta, 17alpha-dihydroxy-5beta-pregnen-20-one, and 5beta-androstane-3,17-dione). In the MT-induced male phase, however, the abrogation of C(17,20) lyase activity and the concomitant activation of 21alpha-hydroxylase/11beta-hydroxylase resulted in the preferential synthesis of polar 21alpha-hydroxlyated 5beta-pregnans (5beta-pregnan-3beta,17alpha,20beta,21alpha-tetrol and 3beta,20beta,21alpha-trihydroxy-5beta-pregnan-3-one) and corticosteroids (11-deoxycortisol and cortisol). Interestingly, synthesis of these 21alpha-hydroxylated 5beta-pregnans and corticosteroids was uniquely compartmentalized in only testicular tissues of the MT-induced males. This study shows that there is selective activation of specific steroidogenic enzymes in the different sexual phases leading to the synthesis of metabolites that may be involved in regulating sex inversion of the grouper.

17-alpha-Hydroxyprogesterone↗
PubMed2001

Sequence analysis of a fish vitellogenin cDNA with a large phosvitin domain.

Vitellogenins (Vtg) are egg-yolk precursor proteins crucial for reproductive success in oviparous animals. We have cloned the first complete cichlid Vtg cDNA from the tilapia fish, Oreochromis aureus. This cDNA has the largest phosvitin (PV) domain amongst piscine Vtgs, being comparable to those of lamprey, Xenopus, and chicken. Thus, the size of PV is independent of the evolutionary advancement of a species. The closer interspecific relationship between O. aureus Vtg1 and Fundulus VtgII than the intraspecific relationship between Fundulus VtgI and II isoforms suggests that teleost ancestors had at least two Vtg isoforms. Contrary to the results of previous phylogenetic inference using Vtgs which indicate that insect lineage is most diverged and nematodes are closer to vertebrate lineage, our results show that nematodes and hexapods form two monophyletic sister groups. Another arthropod taxon, represented by a malacostracan crustacean, Penaeus japonicus, appears to be more closely related to the vertebrates than the hexapods.

Amino Acid Sequence↗
PubMed2001

Roles of an endogenous serum lectin in the immune protection of blue gourami, Trichogaster trichopterus (Pallus) against Aeromonas hydrophila.

The serum of blue gourami, Trichogaster trichopterus (Pallus), contains a calcium-dependent, N-acetyl-galactosamine-binding lectin (BGL) which efficiently activates and enhances the non-specific immune response of fish towards a virulent strain of Aeromonas hydrophila. In the in vitro studies, a lectin concentration range of 0.05-1.0 ng ml(-1) was found to significantly promote phagocytic uptake of the bacteria by macrophages. This effect was further augmented when purified lectin was combined with laminarin (beta-1,3-D-glucan). Supernatants obtained from these lectin-stimulated macrophage cultures also exhibited significant bacteria-killing activities. In addition, complement from naive fish serum, in the presence of purified BGL, was able to kill A. hydrophila. Finally, challenge experiments demonstrated that BGL could confer effective immune protection to naive blue gourami against an Aeromonas infection.

Aeromonas hydrophila↗
PubMed2001

Ontogeny of alpha-amylase gene expression in sea bass larvae (Lates calcarifer).

The enzymatic activities of alpha-amylase and its corresponding messenger RNA levels in developing sea bass (Lates calcarifer) larvae were studied from hatching until 27 days post hatching (dph). An increasing activity of amylase enzyme was measured until 5 dph, and the activity gradually decreased thereafter and reached a constant level by 12 dph. To achieve a better understanding of the molecular mechanisms underlying amylase expression, we have cloned and sequenced a 318-bp fragment of alpha-amylase complementary DNA. Based on this sequence, a real-time reverse transcriptase polymerase chain reaction technique to monitor the changes in the mRNA levels in the larvae was developed. A correlation between enzymatic activity and mRNA level of alpha-amylase could be demonstrated during the early development of sea bass larvae. This suggests that the changes in alpha-amylase are controlled at least at the transcriptional level during early larval development of sea bass.

Journal Article↗
PubMed2001

The structural identifiability and parameter estimation of a multispecies model for the transmission of mastitis in dairy cows.

A structural identifiability analysis is performed on a mathematical model for the coupled transmission of two classes of pathogen. The pathogens, classified as major and minor, are aetiological agents of mastitis in dairy cows that interact directly and via the immunological reaction in their hosts. Parameter estimates are available from experimental data for all but four of the parameters in the model. Data from a longitudinal study of infection are used to estimate these unknown parameters. A novel approach and application of structural identifiability analysis is combined in this paper with the estimation of cross-protection parameters using epidemiological data.

Animals↗
PubMed2001

Electron microscopic observations of a marine fish iridovirus isolated from brown-spotted grouper, Epinephelus tauvina.

The morphogenesis and the ultrastructure of a marine fish iridovirus isolated from diseased grouper, Epinephelus tauvina were studied by electron microscopy. The virus was grown on a marine fish cell line (GP) at 25 degrees C. After appearance of advanced cytopathic effect (CPE), various morphogenetic stages of virus amplification, maturation and assembly were detected in the cytoplasm of virus-infected cells. The matured nucleocapsids were probably formed by insertion of electron-dense core material into a partly forming empty capsid just before completely sealed. The nucleocapsids were located at the assembly sites as pseudocrystalline arrays or scattered individually. In the late phase of infection, the nucleocapsids were enveloped and released by budding from the plasma membrane. The budding virus particles could directly enter neighbouring cells by endocytosis to start the next round infection. Ultrastructure of the grouper iridovirus was studied using the methods of enzymatic digestions and detergent degradations. The purified iridovirus particles showed a three-layered membrane including an external lipoprotein envelope, an inner periodic protein capsid and a lipid-containing membrane. The regular array of surface capsid subunits was observed after degradation with detergent.

Animals↗