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Biomedical subjects

T J Lam

Publications and source records attributed to T J Lam.

At least 37 records · Page 2Linked to original sources
PubMed2001

A multispecies model for the transmission and control of mastitis in dairy cows.

Mastitis in dairy cows is a significant economic and animal welfare issue in the dairy industry. The bacterial pathogens responsible for infection of the mammary gland may be split into two main categories: major and minor pathogens. Infection with major pathogens generally results in clinical illness or strong inflammatory responses and reduced milk yields, whereas minor pathogen infection is usually subclinical. Previous investigations have considered the transmission of these pathogens independently. Experimental evidence has shown cross-protection between species of pathogens. In this study a mathematical model for the coupled transmission of major and minor pathogens along with their interaction via the host was developed in order to consider various methods for controlling the incidence of major pathogen infection. A stability analysis of the model equilibria provides explanations for observed phenomena and previous decoupled modelling results. This multispecies model structure has provided a basis for quantifying the extent of cross-protection between species and assessing possible control strategies against the disease.

Animals↗
PubMed2000

Corticosteroid biosynthesis in vitro by testes of the grouper (Epinephelus coioides) after 17alpha-methyltestosterone-induced sex inversion.

This study reports the unique compartmentalization of cortisol and 11-deoxycortisol biosynthesis in vitro from [(3)H]17alpha-hydroxyprogesterone (17P) in testicular tissues of groupers after sex inversion induced by 17alpha-methyltestosterone (MT). Before MT implantation, the ovarian tissues produced only nonpolar metabolites. Following sex inversion some 6 months later, synthesis of these nonpolar metabolites was not detectable. Instead, cortisol and 11-deoxycortisol, with yields of about 3% and 14%, respectively, were synthesized together with two other polar metabolites. The corticosteroids and polar metabolites were distinctly nondetectable in ovarian tissues of the control fish throughout the experiment. While the significance of this testicular synthesis of corticosteroids is presently unclear, it could be related to the increased energy demands arising from the reorganization of gonadal tissues during sex inversion.

Animals↗
PubMed2000

Adhesion and invasion of Escherichia coli from single and recurrent clinical cases of bovine mastitis in vitro.

Seven strains of Escherichia coli, originating from clinical cases of bovine mastitis, and one Salmonella typhimurium control strain were tested for their ability to adhere to, and invade, bovine mammary epithelial cells (MAC-T cells) in vitro. Four of the seven strains were isolated from cows with chronic intramammary infections with recurrent episodes of clinical mastitis and three strains were isolated from single cases of clinical mastitis. Both adhesion and invasion of all strains were dose and time dependent. The four E. coli strains isolated from recurrent cases of clinical mastitis invaded twice as frequently as and three times faster than the strains isolated from single cases of clinical mastitis. By contrast, there was no difference in the amount or speed of adhesion between the two types of strains of E. coli. Adhesion and invasion curves of E. coli resembled a two-step chain reaction, where invasion was the rate-limiting step. Although adhesion and invasion of E. coli has not been demonstrated in vivo yet, the results of the present study may contribute to an understanding of the pathogenesis of chronic intramammary infections caused by E. coli.

Animals↗
PubMed2000

Isolation and characterisation of a serum lectin from blue gourami, Trichogaster trichopterus (Pallus).

A novel lectin, designated BGL, has been purified from the serum of blue gourami, Trichogaster trichopterus, with the use of (NH4)2SO4 fractionation, affinity chromatography and gel filtration chromatography. Electrophoretic analyses and mass spectrometric study of purified BGL showed that the lectin is composed of two isoforms with native molecular masses estimated to be 65 and 66 kDa, and two subunits of 32 and 34 kDa on SDS-PAGE under non-reducing conditions. Upon reduction with 20 mM dithiothreitol (DTT), BGL showed two close bands of 27 and 29 kDa. After isoelectric focusing, the lectin focused as close double bands at pH 5.6. The N-termini of both isoforms share the same sequence (HGEENRXGPR) and show no significant homology with any known proteins. The BGL agglutinating activity is specifically inhibited by N-acetyl-D-galactosamine and N-acetyl-D-glucosamine, and to a lesser degree by D-(+)-mannose, but not by D-(+)-galactose, D-(+)-glucose, maltose or N-acetyl-D-mannosamine. Haemagglutination assay showed that BGL is more specific for rabbit than mouse, chicken, rat or guinea pig erythrocytes, and haemagglutination was Ca2+-dependent. In addition, BGL could agglutinate a range of micro-organisms and yeast cells, with the exception of some fish pathogens, such as Aeromonas hydrophila (strains: PPD 134/91 and PPD 11/90) and Vibrio harveyi (strain: W618). Localisation of BGL by fluorescein isothiocyanate (FITC)-labelled antibodies revealed that the lectin is associated with the cell surface of fish leukocytes.

Agglutination↗
PubMed1999

Temperature dependence of estrogen binding: importance of a subzone in the ligand binding domain of a novel piscine estrogen receptor.

The full length estrogen receptor from Oreochromis aureus (OaER) was cloned and expressed in vitro and in vivo as a functional transcription factor. Amino acid residues involved in the thermal stability of the receptor are located at/near subzones beta1 and beta3, which are highly conserved in other non-piscine species but not in OaER. Hormone binding studies, however, indicate that OaER is thermally stable but exhibited a approximately 3-fold reduced affinity for estrogen at elevated temperatures. Transfection of OaER into various cell lines cultured at different temperatures displayed a significant estrogen dose-response shift compared with that of chicken ER (cER). At 37 degrees C, OaER requires approximately 80-fold more estrogen to achieve half-maximal stimulation of CAT. Lowering of the incubation temperature from 37 degrees C to 25 degrees C or 20 degrees C resulted in a 4-fold increase in its affinity for estrogen. The thermally deficient transactivation of OaER at temperatures above 25 degrees C was fully prevented by high levels of estrogen. Thus, compared to cER, the OaER exhibits reduced affinity for estrogen at elevated temperature as reflected in its deficient transactivation capability. Amino acid replacements of OaER beta3 subzones with corresponding amino acids from cER could partially rescue this temperature sensitivity. The three-dimensional structure of the OaER ligand binding domain (LBD) was modelled based on conformational similarity and sequence homology with human RXRalpha apo, RARgamma holo and ERalpha LBDs. Unliganded and 17beta-estradiol-liganded OaER LBD retained the overall folding pattern of the nuclear receptor LBDs. The residues at/near the subzone beta3 of the LBD constitute the central core of OaER structure. Thus, amino acid alteration at this region potentially alters the structure and consequently its temperature-dependent ligand binding properties.

Amino Acid Sequence↗
PubMed1999

Synergistic effects of nuclear factors--GATA, VBP and ER in potentiating vitellogenin gene transcription.

The Oreochromis aureus vitellogenin (OaVtg) gene contains three imperfect oestrogen response elements (EREs) and GATA and VBP (vitellogenin binding protein) binding sites. An analysis of the promoter indicates that the 5'-flanking region up to position -625 is sufficient to mediate E(2) control. Furthermore, transfection of deletion and mutagenised promoters indicates that both GATA and VBP synergise with ER, and thus contribute to the regulation of the endogenous OaVtg gene. These findings support the notion that the interplay of promoter elements mediates proper hormone-dependent and tissue-specific expression of the OaVtg gene, regardless of non-consensus sequence context of EREs and VBP.

Animals↗
PubMed1999

Quarter-milk somatic cell count at calving and at the first six milkings after calving.

Thirty cows were studied during the first six milkings after calving. Quarter foremilk samples were collected by the farmers at calving and at six subsequent milkings. Geometric-mean somatic cell count (SCC) decreased from 593,000 at calving to 126,000 cells/ml at the sixth milking after calving. In quarters infected with major pathogenic bacteria, geometric-mean SCC was 3,229,000 cells/ml at calving, and 1,257,000 cells/ml at the sixth milking after calving. In quarters infected with minor pathogenic bacteria, geometric-mean SCC was 1,000,000 cells/ml at calving, and 170,000 cells/ml at the sixth milking after calving. In culture-negative quarters, geometric-mean SCC decreased from 306,000 at calving to 42,000 cells/ml at the sixth milking after calving. Quarter SCC can be used early postpartum to give an indication of intra-mammary infection status.

Animals↗
PubMed1999

Faithful expression of green fluorescent protein (GFP) in transgenic zebrafish embryos under control of zebrafish gene promoters.

Although the zebrafish has become a popular model organism for vertebrate developmental and genetic analyses, its use in transgenic studies still suffers from the scarcity of homologous gene promoters. In the present study, three different zebrafish cDNA clones were isolated and sequenced completely, and their expression patterns were characterized by whole-mount in situ hybridization as well as by Northern blot hybridization. The first clone encodes a type II cytokeratin (CK), which is specifically expressed in skin epithelia in early embryos and prominently expressed in the adult skin tissue. The second clone is muscle specific and encodes a muscle creatine kinase (MCK). The third clone, expressed ubiquitously in all tissues, is derived from an acidic ribosomal phosphoprotein P0 (arp) gene. In order to test the fidelity of zebrafish embryos in transgenic expression, the promoters of the three genes were isolated using a rapid linker-mediated PCR approach and subsequently ligated to a modified green fluorescent protein (gfp) reporter gene. When the three hybrid GFP constructs were introduced into zebrafish embryos by microinjection, the three promoters were activated faithfully in developing zebrafish embryos. The 2.2-kb ck promoter was sufficient to direct GFP expression in skin epithelia, although a weak expression in muscle was also observed in a few embryos. This pattern of transgenic expression is consistent with the expression pattern of the endogenous cytokeratin gene. The 1.5-kb mck promoter/gfp was expressed exclusively in skeletal muscles and not elsewhere. By contrast, the 0.8-kb ubiquitous promoter plus the first intron of the arp gene were capable of expressing GFP in a variety of tissues, including the skin, muscle, lens, neurons, notochord, and circulating blood cells. Our experiments, therefore, further demonstrated that zebrafish embryos can faithfully express exogenously introduced genes under the control of zebrafish promoters.

Animals↗
PubMed1999

Identification of differentially expressed genes in Con A-activated carp (Cyprinus carpio L.) leucocytes.

A cDNA library was constructed from the message RNA (mRNA) obtained from Con A-induced head kidney (HK) leucocytes of carp (Cyprinus carpio L.). Differential screening of the cDNA was carried out by hybridization against the total cDNA probes from normal, Con A-uninduced HK leucocytes or Con A-induced HK leucocytes of carp. The differential expression patterns of certain cDNA clones were confirmed by Southern-blot and Northern-blot analysis. Single-pass of the sequencing analysis and homology search in Genbank (EMBL) revealed those differentially expressed cDNA clones encode for cytochrome c oxidase sub-unit II and III (COII and COIII), elongation factor-1 beta (EF-1 beta), bleomycin hydrolase (BH), heat shock cognate protein 70 (HSC70) and 16S ribosomal RNA (16S rRNA).

Amino Acid Sequence↗
PubMed1999

Fast skeletal muscle-specific expression of a zebrafish myosin light chain 2 gene and characterization of its promoter by direct injection into skeletal muscle.

A zebrafish myosin light chain 2 cDNA clone was isolated and characterized. Sequence analysis of the clone revealed a high homology with the mammalian and avian genes encoding the fast skeletal muscle isoform, MLC2f. In situ hybridization and Northern blot hybridization analyses indicated that the zebrafish MLC2f mRNA is expressed exclusively in the fast skeletal muscle. Ontogenetically, the MLC2f mRNA appears around 16 hours postfertilization (hpf) in the first few well-formed anterior somites. At later stages, the MLC2f mRNA can also be detected in fin buds, eye muscles, and jaw muscles. To develop a useful model system for analyzing muscle gene regulation, the promoter of the zebrafish MLC2f gene was isolated and linked to the chloramphenicol acetyltransferase (CAT) reporter gene. The MLC2f/CAT chimeric constructs were analyzed by direct injection into the zebrafish skeletal muscle, and significant CAT activity was observed; in contrast, little or no CAT activity was generated from a similarly injected prolactin gene promoter/CAT gene construct. Within the 1 kb of the MLC2f promoter region, several MEF2-binding sites and E-boxes were identified, suggesting that MLC2f can be regulated by muscle transcription factors MEF2 and myogenic bHLH proteins. A 5' deletion analysis indicated that the proximal 79 nucleotides from the transcription start site, which contains a single MEF2-binding site, is sufficient to drive a high level of CAT activity in injected muscle. Internal deletion of the MEF2 element in the -79-bp construct caused an 80% decrease in CAT activity, whereas internal deletion of the same MEF2 element in a -1044-bp construct had no effect on induced CAT activity. These observations suggest that an MEF2 element is important to activate the MLC2f gene in muscle cells, and the effect of loss of the proximal MEF2 element can be compensated for by the presence of the upstream MEF2 elements. This study also demonstrated that direct injection of DNA into skeletal muscle is a valid and valuable approach to analyze muscle gene promoters in the zebrafish.

Amino Acid Sequence↗
PubMed1999

The mudskipper, Periophthalmodon schlosseri, actively transports NH4+ against a concentration gradient.

Periophthalmodon schlosseri can maintain ammonia excretion rates and low levels of ammonia in its tissues when exposed to 8 and 30 mM NH4Cl, but tissue ammonia levels rise when the fish is exposed to 100 mM NH4Cl in 50% seawater. Because the transepithelial potential is not high enough to maintain the NH4+ concentration gradient between blood and water, ammonia excretion under such a condition would appear to be active. Branchial Na+-K+-ATPase activity is very high and can be activated by physiological levels of NH4+ instead of K+. Ammonia excretion by the fish against a concentration gradient is inhibited by the addition of ouabain and amiloride to the external medium. It is concluded that Na+-K+-ATPase and an Na+/H+ exchanger may be involved in the active excretion of ammonia across the gills. This unique ability of P. schlosseri to actively excrete ammonia is related to the special structure of its gills and allows the fish to continue to excrete ammonia while air exposed or in its burrow.

Ammonia↗
PubMed1999

Recurrent clinical mastitis caused by Escherichia coli in dairy cows.

In this study, the occurrence of persistent intramammary infections caused by Escherichia coli with recurrent episodes of clinical mastitis caused by E. coli are described for a cohort of 300 Dutch dairy herds. Calculations on the recurrent episodes were based on data collected by dairy farmers. The genotype of the E. coli strains was determined by means of a polymerase chain reaction using enterobacterial repetitive intergenic consensus (ERIC) primers, resulting in a DNA fingerprint. Quarters in which the same E. coli genotype was found were considered to be persistently infected. In 4.77% of all episodes of clinical mastitis caused by E. coli, persistent intramammary infections caused by the same E. coli genotype were found. Based on the occurrence of the same genotypes, we concluded that, in 2.98% of all episodes, transmission of E. coli strains among quarters within one cow might have occurred. In 13.04% of all episodes of clinical mastitis caused by E. coli in the study, different E. coli genotypes were isolated from recurrent episodes of clinical mastitis within the same cow, indicating that these cows were highly susceptible to recurrent intramammary infections caused by E. coli.

Animals↗
PubMed1999

Management practices associated with the incidence rate of clinical mastitis.

Risk factors for the incidence rate of clinical mastitis were studied in 274 Dutch dairy herds. Variables that were associated with resistance to disease were the feeding, housing, and milking machine factors. Variables that were associated with exposure were grazing, combined housing of dry cows and heifers, and calving area hygiene. Postmilking teat disinfection in herds with a low bulk milk somatic cell count and years of practicing dry cow therapy were positively associated with the incidence rate of clinical mastitis. Herds with a low bulk milk somatic cell count and in which postmilking teat disinfection was not used had lower incidence rates of clinical mastitis than did other herds. The incidence rate of clinical mastitis caused by Escherichia coli was mostly related to housing conditions, hygiene, and machine milking. The incidence rate of clinical mastitis caused by Staphylococcus aureus was mostly related to factors associated with bulk milk somatic cell count and factors that might be due to cause and effect reversal. A strong positive correlation existed between the incidence rate of clinical mastitis caused by Streptococcus dysgalactiae and the incidence rate of clinical mastitis caused by Staph. aureus. The incidence rate of clinical mastitis caused by Streptococcus dysgalactiae was related to nutrition, milking technique, and machine milking. The incidence rate of clinical mastitis caused by Streptococcus uberis was associated with factors related to housing, nutrition, and machine milking.

Animal Nutritional Physiological Phenomena↗
PubMed1999

Management style and its association with bulk milk somatic cell count and incidence rate of clinical mastitis.

Management style and its association with bulk milk somatic cell count (SCC) and the incidence rate of clinical mastitis were studied in 300 Dutch dairy herds. Cluster analysis was used to identify groups of farmers who had similar management styles for the prevention of mastitis. Two groups of farmers could be differentiated. The management style of the first group of farmers was described as clean and accurate; the management style of the second group of farmers was described as quick and dirty. The relationship between clusters and the bulk milk SCC category was high. The relationship between clusters and incidence rate of clinical mastitis was weak. Compared with herds with a high (250,000 to 400,000 cells/ml) bulk milk SCC, herds with a low bulk milk SCC (< or = 150,000 cells/ml) were managed by farmers who were younger, had children with a higher education, and were more eager to invest. Farmers of herds with a low bulk milk SCC kept better records and were more familiar with each cow in their herds. The most striking difference between farmers of herds with low and high bulk milk SCC was that the first group worked precisely rather than fast; the latter group of farmers worked quickly rather than precisely. As a result, the farms with herds that had a low bulk milk SCC had better hygienic conditions than those farms with herds that had a high bulk milk SCC. We also discuss the implications for producer education with regard to udder health.

Age Factors↗
PubMed1998

A novel piscine vitellogenin gene: structural and functional analyses of estrogen-inducible promoter.

The Oreochromis aureus vitellogenin, OaVtg, gene spans 9 kb and contains 34 exons. Its transcription start site is located 15 bp upstream of the translational start codon. Although the OaVtg promoter has a nonconsensus TATA, transient transfection assay showed that this promoter is capable of driving basal transcription. Two imperfect estrogen response elements: EREp (proximal) and EREd (distal) are located in the promoter at - 532 and - 1352, respectively. In competition gel mobility-shift assays, only EREp exhibited specific binding of the recombinant estrogen receptor protein, GST-C/D OaER. Another imperfect ERE (EREexon2) was detected within exon 2 of the OaVtg gene. This is a novel finding for a vitellogenin (Vtg) gene. EREexon2 similarly showed specific recognition of GST-C/D OaER. Both EREp and EREexon2 showed comparable binding affinities as consensus ERE. In transient transfections, the OaVtg promoter, EREp and EREd elicited significant increase in estrogen-dependent synthesis of CAT protein. Hence, we propose that the non-consensus OaVtg EREs contribute to the estrogen-dependent regulation of the OaVtg gene in vivo.

Amino Acid Sequence↗
PubMed1998

Cloning and characterization of vascular endothelial growth factor (VEGF) from zebrafish, Danio rerio.

We have cloned and sequenced a zebrafish (Danio rerio) Vascular Endothelial Growth Factor (vegf) cDNA. It encodes a precursor protein of 188 amino acids with a putative 23 amino acids signal peptide. Sequence comparison analysis indicates that the zebrafish vegf cDNA corresponds to the human VEGF165 isoform and shows about 52% identity to human VEGF165 at the amino acid level. A 2.8 kb vegf message RNA was detected in adult zebrafish by Northern blot analysis. Expression of vegf165 is also detected by RT-PCR in adult fish and throughout the zebrafish embryonic development. Whole mount in situ hybridization of zebrafish embryos indicates strong expression in four areas of the 18-19 h post-fertilization (hpf) embryo: within the anterior central nervous system in the prospective optic stalk, in mesoderm overlapping the bilaterally located merging heart fields, in mesoderm underlying and flanking the hindbrain posterior to rhombomere 4, and in medial regions of the somites. The study of vegf function in zebrafish embryonic vascular development will contribute to our understanding of the mechanisms of vertebrate endothelial cell differentiation and vasculature formation.

Amino Acid Sequence↗
PubMed1998

Synthesis of 17,20alpha/beta-dihydroxy-4-pregnen-3-one and 5beta-pregnanes in spermatozoa of primary and 17alpha-methyltestosterone-induced secondary male grouper (Epinephelus coioides).

The in vitro metabolism of [3H]17-hydroxyprogesterone by spermatozoa of primary and 17alpha-methyltestosterone (MT)-induced secondary male grouper (Epinephelus coioides) has been examined. With only the labeled precursor, the four predominant 5beta-pregnanes (17, 20alpha-dihydroxy-5beta-pregnan-3-one; 5beta-pregnan-3alpha,17, 20alpha-triol; 3alpha,17-dihydroxy-5beta-pregnan-20-one; and 17hydroxy-5beta-pregnan-3,20-dione) accounted for 75% of the total product yield, with the remainder comprising 17, 20alpha-dihydroxy-4-pregnen-3-one (17,20alphaP) and traces (3%) of 17,20beta-dihydroxy-4-pregnen-3-one. This is the first report of the synthesis of these 5beta-pregnanes by teleost spermatozoa. The addition of excess unlabeled precursor caused a marked shift to the synthesis of only 17,20alphaP. The steroidogenic profile in spermatozoa of the primary and secondary males appears similar in both cases. The shift to 17,20alphaP synthesis at high substrate concentration may suggest a gonadotropin (GtH) control of 17, 20alphaP synthesis, involving the competition for 17P between the high-activity, low-capacity 5beta-reductases and the low-activity, high-capacity 20alpha-HSD. Poor spermiation in MT-induced males may thus be due to the lack of 17P arising from low GtH secretion in vivo in the secondary males.

17-alpha-Hydroxyprogesterone↗