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Biomedical subjects

J D Brooker

Publications and source records attributed to J D Brooker.

At least 19 recordsLinked to original sources
PubMed2006

A fermentation assay to evaluate the effectiveness of antimicrobial agents on gut microflora.

The measurement of gas produced as a fermentation end product in vitro was correlated with absorbance as a measure of bacterial growth and was used as a rapid screening procedure to test the antimicrobial activity of certain essential oil and tannin secondary plant metabolites on gastrointestinal microorganisms from chickens. The assay was optimised using Clostridium perfringens and Lactobacillus fermentum, and tested in antimicrobial assays against C. perfringens; the minimum inhibitory concentration for each essential oil and condensed tannin was determined. The effect of penicillin-G on C. perfringens, in both growth and fermentation assays, was similar, and for all secondary metabolites tested, concentrations that inhibited fermentation were also bacteriocidal. The assay was also used to demonstrate the effect of dietary composition and enzyme supplementation on fermentation of mixed gut microflora in vitro; results are compared with in vivo results for the same dietary treatments. The data demonstrate that the effects of bioactive secondary plant products and feed composition on individual organisms or mixed gut microflora can be tested by analysis of fermentative activity in vitro, and that this provides a rapid assay for testing potential poultry feed additives before in vivo trials.

Animals↗
PubMed2005

Biochemistry of plant secondary metabolites and their effects in animals.

Plant secondary metabolites, which include a wide variety of phytochemicals, have always been constituents of the diets of man and other animals. Although a high proportion of these phytochemicals have been considered to be of little value in plants (although this view is changing), they have frequently been shown to have adverse effects on animals when ingested. The effects depend to a great extent on the chemistry of the compounds, their concentration in the diet and the amount consumed, and are further dependent on the health status of the animals. Traditionally, most studies of the effects of these compounds on animals have focused on their adverse effects and how to alleviate them. However, recent public concern about the use of synthetic compounds in animal diets to enhance performance and health and welfare issues, coupled with changes in regulations on the use of synthetic medicaments, has stimulated interest and research in the use and effects of phytochemicals in the diets of farmed animals. Phytochemicals vary in their chemistry but can be divided into hydrophilic and hydrophobic compounds, of which a wide variety of polyphenolic and terpenoid compounds, as well as alkaloids, carbohydrates and non-protein amino acids, invoke special interest. The chemistry, biochemistry and mechanisms of action of these compounds in plants and their effects in animals when ingested will be explored.

Animal Feed↗
PubMed2001

Effect of hydrolysable and condensed tannins on growth, morphology and metabolism of Streptococcus gallolyticus (S. caprinus) and Streptococcus bovis.

Streptococcus gallolyticus (S. caprinus) was resistant in vitro to at least 7% (w/v) tannic acid and 4% (w/v) acacia condensed tannin, levels 10-fold greater than those tolerated by S. bovis. Growth of S. gallolyticus in liquid medium was characterized by a lag period which increased, and a growth rate which decreased, with increasing tannin concentration. S. gallolyticus was also more tolerant to the presence of simple phenolic acid monomers than was S. bovis, but the lag period was still concentration dependent. Gallate decarboxylase activity in S. gallolyticus was elevated in the presence of tannic acid or gallic acid but not with other phenolic acids. Scanning electron microscopic analysis showed that both the size and shape of S. gallolyticus and S. bovis changed in response to tannin but only S. gallolyticus was surrounded by an extracellular polysaccharide matrix which accumulated in a tannin-concentration-dependent fashion. Washing of the cells to remove extracellular polysaccharide increased the lag period of S. gallolyticus in the presence of 1% (w/v) tannic acid from 4 h to 6 h. In contrast, increasing extracellular polysaccharide synthesis in S. bovis did not increase its tolerance to tannic acid. These data demonstrate that S. gallolyticus has developed a number of mechanisms to reduce the potential effect of tannins on cell growth, and that these mechanisms provide the organism with a selective advantage over S. bovis when grown in the presence of tannins.

Carboxy-Lyases↗
PubMed1997

Gene knockout of the intracellular amylase gene by homologous recombination in Streptococcus bovis.

Streptococcus bovis expresses two different amylases, one intracellular and the other secreted. A suicide vector containing part of the intracellular alpha-amylase gene from Streptococcus bovis WI-1 was recombined into the S. bovis WI-1 chromosome to disrupt the endogenous gene. Recombination was demonstrated by Southern blot, and zymogram analysis confirmed the loss of the intracellular amylase. Amylase activity in cell-free extracts of the recombinant grown in the presence of 1% starch was only 7% of wild type. The rate of logarithmic growth of the recombinant was 15-20% of the wild type in medium containing either 1% glucose, starch, or cellobiose. Revertants and non-amylase control recombinants had logarithmic growth rates that were the same as wild type. Plasmid transformants containing multiple copies of the cloned gene expressed up to threefold higher levels of intracellular amylase activity than wild type but did not demonstrate elevated growth rates. These results suggest that a critical level of expression of the intracellular amylase gene may be important for rapid growth of the bacterium.

Amylases↗
PubMed1995

Characterization of tannin acylhydrolase activity in the ruminal bacterium Selenomonas ruminantium.

A strain of the anaerobe Selenomonas ruminantium subsp. ruminantium that is capable of growing on tannic acid or condensed tannin as a sole energy source has been isolated from ruminal contents of feral goats browsing tannin-rich Acacia sp. Growth on tannic acid was accompanied by release of gallic acid into the culture medium but the bacterium was incapable of using gallic acid as a sole energy source. Tannin acylhydrolase (EC 3.1.1.20) activity was measured in crude cell-free extracts of the bacterium. The enzyme has a pH optimum of 7, a temperature optimum of 30-40 degrees C and a molecular size of 59 kDa. In crude extracts, the maximal rate of gallic acid methyl ester hydrolysis was 6.3 micromol min(-1) mg(-1) of protein and the K(m) for gallic acid methyl ester was 1.6 mM. Enzyme activity was displayed in situ in polyacrylamide and isoelectric focusing gels and was demonstrated to increase 17-fold and 36-fold respectively when cells were grown in the presence of gallic acid methyl ester or tannic acid.

Journal Article↗
PubMed1995

A defined medium for rumen bacteria and identification of strains impaired in de novo biosynthesis of certain amino acids.

A completely defined growth medium has been developed to determine the nitrogen requirements for several species of ruminal bacteria, and has revealed two strains which are impaired in de novo biosynthesis of certain amino acids. Using NH4Cl as a sole nitrogen source, the medium supported growth of Butyrivibrio, Selenomonas, Prevotella and Streptococcus species. One strain of B. fibrisolvens (E14) and one strain of P. ruminicola (GA33) did not grow in the presence of NH4Cl until the medium was supplemented with amino acids or peptides. For B. fibrisolvens strain E14, methionine was identified as the specific growth-limiting amino acid although methionine alone did not support growth in the absence of NH4Cl. For P. ruminicola strain GA33, any individual amino acid other than methionine or cysteine could supplement the medium and support growth. Enzyme assays confirmed a lack of NADH and NADPH-dependent glutamate dehydrogenase (GDH) activities in this strain.

Amino Acids↗
PubMed1995

A gene-targeting suicide vector for Streptococcus bovis.

A gene-targeting suicide vector for Streptococcus bovis has been constructed using the Escherichia coli/Streptococcus shuttle plasmid, pMU1328, and a region derived from the broad host-range, Gram-positive transposon, Tn916. This suicide plasmid replicates autonomously in E. coli, but not in Strep. bovis or Strep. bovis Tn916. Under positive selection, the plasmid was shown to integrate into Strep. bovis Tn916 chromosomal DNA at a frequency of 3 x 10(-8) cell-1 and was stably maintained for at least 100 generations in the absence of selection. This is the first report of a recombination system in ruminal bacteria. The ability to target genes, knock out specific functions or introduce novel genes into these micro-organisms will allow ruminal species to be manipulated and may eventually lead to improved animal production.

Bacterial Proteins↗
PubMed1993

Characterization, sequence, and replication of a small cryptic plasmid from Selenomonas ruminantium subspecies lactilytica.

A 2.5-kb cryptic plasmid, pJDB21, from the gram-negative ruminal anaerobe, Selenomonas ruminantium subspecies lactilytica, was mapped and sequenced. Five open reading frames (ORFs) were predicted and expression of two ORFs was demonstrated. Analysis of the predicted amino acid sequence of the ORF 1 protein indicated approximately 30% homology with the replication protein (rep) common to many gram-positive plasmids, and a highly conserved sequence representing the origin of replication in these plasmids was located upstream of ORF 1. This finding was consistent with a rolling circle form of replication for pJDB21. Transformation of Escherichia coli K-12 UB1636pol Ats with pJDB21 showed that the plasmid replicated independently of DNA polymerase I and produced a single-stranded DNA intermediate. Deletion analyses localized the E. coli replication function to a 1.4-kb sequence that was mapped to the predicted rep gene.

Amino Acid Sequence↗
PubMed1992

Complete nucleotide sequence of a Selenomonas ruminantium plasmid and definition of a region necessary for its replication in Escherichia coli.

A plasmid from Selenomonas ruminantium subspecies lactilytica has been subcloned in Escherichia coli K-12 and completely sequenced. Three open reading frames (ORFs) of 909, 801, and 549 bp were identified and the complete sequence was analyzed by comparison with DNA and protein databases. No significant deoxynucleotide or amino acid sequence homology with other published genes or proteins was detected. The plasmid was shown to replicate independently in E. coli K-12 by a DNA polymerase I-dependent mechanism and deletion analysis defined the DNA sequence responsible for this phenotype.

Amino Acid Sequence↗
PubMed1992

Improved animal production by genetic engineering of ruminal bacteria.

Ruminant production is a major focus of Australian agriculture. The ability of ruminant animals such as sheep and cattle to make productive use of low quality plant materials depends on the activity and efficiency of the anaerobic microbial population that resides in the rumen. Factors that affect ruminant production include the ability of cellulolytic microorganisms to digest plant structural polysaccharides (primarily cellulose and hemicellulose), the capacity of microorganisms to metabolise and detoxify otherwise inhibitory plant products and the efficiency of nitrogen utilisation by ruminal organisms. This review will consider some current Australian research programs aimed at improving ruminant production efficiency by genetic engineering of ruminal bacteria.

Agriculture↗
PubMed1990

Monoclonal antibodies against the ruminal bacterium Selenomonas ruminantium.

Monoclonal antibodies were raised against whole cells of two different strains of Selenomonas ruminantium and tested for specificity and sensitivity in immunofluorescence and enzyme-linked immunosorbent assay procedures. Species-specific and strain-specific antibodies were identified, and reactive antigens were demonstrated in solubilized cell wall extracts of S. ruminantium. A monoclonal antibody-based solid-phase immunoassay was established to quantify S. ruminantium in cultures or samples from the rumen, and this had a sensitivity of 0.01 to 0.02% from 10(7) cells. For at least one strain, the extent of antibody reaction varied depending upon the stage of bacterial growth. Antigen characterization by immunoblotting shows that monoclonal antibodies raised against two different strains of S. ruminantium reacted with the same antigen on each strain. For one strain, an additional antigen reacted with both monoclonal antibodies. In the appropriate assay, these monoclonal antibodies may have advantages over gene probes, both in speed and sensitivity, for bacterial quantification studies.

Animals↗
PubMed1988

Use of a unique gene sequence as a probe to enumerate a strain of Bacteroides ruminicola introduced into the rumen.

Cloned fragments of genomic DNA from the ruminal anaerobe Bacteroides ruminicola subsp. brevis B14 were isolated and used as hybridization probes to identify closely related bacterial species. One DNA fragment unique to strain B14 was tested to determine its sensitivity in detecting homologous sequences among total ruminal microbial DNA. In a DNA titration experiment, the probe was capable of detecting strain B14 sequences in vitro down to 0.1% of the total bacterial DNA present in a hybridization assay. There was no detectable signal for total ruminal bacterial DNA. The specificity of this DNA fragment was exploited to enumerate strain B14 in a fresh mixed suspension of ruminal bacteria in vitro and after inoculation of the strain into the rumen. In vitro strain B14 had a half-life of 9 h. However, following inoculation into the rumen there was a very rapid loss of the strain to below the detectable limit within 3 h. The half-life was less than 30 min. This loss was not due to ruminal dilution or to bacteriophage attack but was possibly the result of a specific bacteriocinlike activity present in the rumen and detectable in fresh ruminal fluid.

Animals↗
PubMed1988

Isolation and characterization of a temperate bacteriophage from the ruminal anaerobe Selenomonas ruminantium.

A temperate bacteriophage was obtained from an isolate of the ruminal anaerobe Selenomonas ruminantium. Clear plaques that became turbid on further incubation occurred on a lawn of host bacteria. Cells picked from a turbid plaque produced healthy liquid cultures, but these often lysed on storage. Mid-log-phase liquid cultures incubated with the bacteriophage lysed and released infectious particles with a titer of up to 3 X 10(7) PFU/ml. A laboratory strain of S. ruminantium, HD-4, was also sensitive to this bacteriophage, which had an icosohedral head (diameter, 50 nm) and a flexible tail (length, 140 nm). The bacteriophage contained 30 kilobases of linear, double-stranded DNA, and a detailed restriction map was constructed. The lysogenic nature of infection was demonstrated by hybridization of bacteriophage DNA to specific restriction fragments of infected host genomic DNA and by identification of a bacteriophage genomic domain which may participate in integration of the bacteriophage DNA. Infection of S. ruminantium in vitro was demonstrated by two different methods of cell transformation with purified bacteriophage DNA.

Animals↗
PubMed1984

Molecular cloning of hepatic 5-aminolevulinate synthase.

Hepatic 5-aminolevulinate synthase was induced in chick embryos by administration of the porphyrinogenic drugs 2-allyl-2-isopropylacetamide and 3,5-diethoxycarbonyl-1,4-dihydrocollidine. A cDNA library was constructed from drug-induced liver mRNA and clones containing sequences coding for 5-aminolevulinate synthase were identified by hybrid-selected translation. The identity of these clones was confirmed by comparing DNA sequence data with the amino acid sequence of peptides from purified 5-aminolevulinate synthase. From Northern blot analysis the size of the mRNA for 5-aminolevulinate synthase was estimated to be 2800 base pairs, approximately 600 base pairs more than that required to code for the primary translation product of relative molecular mass 74000.

5-Aminolevulinate Synthetase↗
PubMed1983

Hemin inhibits transfer of pre-delta-aminolevulinate synthase into chick embryo liver mitochondria.

Pulse labelling studies in chick embryo livers show that hemin prevents the transfer of drug induced pre-delta-aminolevulinate synthase from the cytosol into the mitochondria, leading to an accumulation of precursor in the cytosol. No effect of hemin was observed on the transfer of pre-pyruvate carboxylase into mitochondria. These results eliminated a general toxic effect of hemin on mitochondrial import of proteins and are consistent with the view that hemin specifically inhibits the transfer of ALA synthase.

5-Aminolevulinate Synthetase↗
PubMed1983

Evidence that 2-allyl-2-isopropylacetamide, phenobarbital and 3,5-diethoxycarbonyl-1,4-dihydrocollidine induce the same cytochrome P450 mRNA in chick embryo liver.

The induction of cytochrome P450 in chick embryo liver has been studied using three different porphyrinogenic drugs, 2-allyl-2-isopropylacetamide, 3,5-diethoxycarbonyl-1,4-dihydrocollidine and phenobarbital. Pulse-labelling studies have shown that for each drug the cytochrome P450 synthesized either in ovo or in a wheat germ translation system reacted immunologically with antibody raised against the purified 2-allyl-2-isopropylacetamide-induced enzyme (Mr = 50000). To investigate whether this is due to the three drugs inducing the same protein or different proteins with common immunological determinants, nucleic acid hybridization studies have been carried out using a recently characterised 2-allyl-2-isopropylacetamide-induced cytochrome P450 cloned cDNA probe [Brooker, J. D. et al. (1982) Eur. J. Biochem. 129, 325-333]. It has been shown that the mRNA induced by each drug hybridizes with this probe and all are of similar size. The melting profile of the mRNA . cDNA hybrids indicates that the mRNAs induced by the three drugs have at least 98% homology with the cDNA probe. Restriction endonuclease digestions of total chick embryo genomal DNA and a chick cytochrome P450 genomal clone indicates that the cytochrome P450 gene homologous with the cDNA probe is represented in the genome only once. These results strongly suggest that the three drugs cause increased levels of the same cytochrome P450 mRNA, possibly due to enhanced expression of the same gene. Results are also presented which show that other cytochrome-P450-inducing drugs, 3-methylcholanthrene, beta-naphthoflavone or pregnenolone-16 alpha-carbonitrile do not increase the level of the 2-allyl-2-isopropylacetamide-inducible mRNA but rather reduce it to a level which was lower than that of the untreated controls.

Acetamides↗