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Biomedical subjects

D J O'Sullivan

Publications and source records attributed to D J O'Sullivan.

At least 19 recordsLinked to original sources
PubMed1999

Nisin independent induction of the nisA promoter in Lactococcus lactis during growth in lactose or galactose.

Nisin biosynthesis is autoregulated extracellularly by the mature and modified peptide. To investigate other regulatory effects on nisin biosynthesis, a transcription fusion of the nisA promoter from Lactococcus lactis ATCC 11454 to the promoterless lacZ gene from Streptococcus thermophilus was constructed. This fusion construct, pDOC99, expressed beta-galactosidase in L. lactis ATCC 11454 growing in M17 medium containing glucose (M17G). Consistent with the known model for transcription of nisA, pDOC99 did not express beta-galactosidase in the non-nisin producer, L. lactis LM0230 grown in M17G, unless the nisRK genes (cloned in pDOC23) were included in trans and nisin was added to the medium. Growth of this strain in M17 containing lactose or galactose, resulted in nisA transcription, even in the absence of exogenous nisin. This expression was independent of pDOC23. Furthermore, nisA transcription in L. lactis LM0230(pDOC99) grown in M17G could be induced by the addition of exogenous galactose, with maximum induction occurring at concentrations > 5 mM.

Galactose↗
PubMed1999

The sydney multicentre study of Parkinson's disease: progression and mortality at 10 years.

OBJECTIVES: To report on a 10 year follow up of patients with idiopathic Parkinson's disease, particularly with respect to mortality and the effect of early treatment with bromocriptine. METHODS: The patients are from the 149 new patients recruited for a double blind, randomised study of low dose levodopa-carbidopa versus low dose bromocriptine. Patients were examined neurologically at least yearly. Neuropsychological examinations were performed at 0, 3, 5, and 10 years. Mortality and cause of death in these patients were compared with the Australian population using standardised mortality ratios (SMRs). Mortality and disease progression were compared by sex and treatment group. Predictors of death within 10 years, nursing home admission, and progression in Columbia score of >/=20 points were examined by logistic regression analysis. RESULTS: Thirteen patients were excluded as having atypical Parkinsonism and six were lost to follow up. All available patients have been followed up for 10 years. Fifty patients (38%) were dead by 10 years and 63 by the last follow up. The SMR was 1.58 for all patients (p<0. 001). There was no significant difference in SMRs between the sexes. The mean duration of disease until death was 9.1 years. Parkinson's disease was thought to have contributed substantially to the death of 30 patients. The most common cause of death was pneumonia. Women progressed at a similar rate to men until 8 years, when the severity of their disease as measured by Hoehn and Yahr stage became greater (p<0.05). Older age of onset correlated with increased risk of death but the SMR was increased even in those aged <70 years (SMR 1.80, p=0.03). Early use of bromocriptine did not reduce mortality or slow progression of disease. One quarter of all patients had been admitted to nursing homes by 10 years. Only four patients were still employed. CONCLUSIONS: Mortality in Parkinson's disease remains increased despite low dose levodopa-carbidopa therapy and no additional benefit was gained from early use of bromocriptine. Duration of disease was similar to that in the era before levodopa.

Age Distribution↗
PubMed1998

Procedure for quantifiable assessment of nutritional parameters influencing nisin production by Lactococcus lactis subsp. lactis.

A modified rapid plate assay procedure was developed, that allowed quantifiable measurement of nisin production by Lactococcus lactis growing directly on agar media. Using this direct plate assay, several nutritional parameters were assessed for their influence on nisin production (as distinct from their influence on growth) by L. lactis subsp. lactis ATCC 11454 growing on standard M17 based media over 3 and 6 h incubation periods. Glucose was found to be the optimal carbon source tested, with glycerol having the greatest suppressive effect. The addition of salts suppressed nisin production on a per cell basis, except MnCl2. This direct plate method proved to be a good pilot assay for rapidly and quantifiably investigating the initial effects of different parameters on nisin production by L. lactis, prior to conducting more intensive broth batch culture assays. The data obtained in this study indicate that certain nutritional parameters can impose a repressive effect on nisin production. Elucidation of how these parameters control the amount of nisin produced will provide further insight into the regulation of nisin biosynthesis in L. lactis.

Biological Assay↗
PubMed1998

A leucine repeat motif in AbiA is required for resistance of Lactococcus lactis to phages representing three species.

The abiA gene encodes an abortive bacteriophage infection mechanism that can protect Lactococcus species from infection by a variety of bacteriophages including three unrelated phage species. Five heptad leucine repeats suggestive of a leucine zipper motif were identified between residues 232 and 266 in the predicted amino acid sequence of the AbiA protein. The biological role of residues in the repeats was investigated by incorporating amino acid substitutions via site-directed mutagenesis. Each mutant was tested for phage resistance against three phages, phi 31, sk1, and c2, belonging to species P335, 936, and c2, respectively. The five residues that comprise the heptad repeats were designated L234, L242, A249, L256, and L263. Three single conservative mutations of leucine to valine in positions L235, L242, and L263 and a double mutation of two leucines (L235 and L242) to valines did not affect AbiA activity on any phages tested. Non-conservative single substitutions of charged amino acids for three of the leucines (L235, L242, and L256) virtually eliminated AbiA activity on all phages tested. Substitution of the alanine residue in the third repeat (A249) with a charged residue did not affect AbiA activity. Replacement of L242 with an alanine elimination phage resistance against phi 31, but partial resistance to sk1 and c2 remained. Two single proline substitutions for leucines L242 and L263 virtually eliminated AbiA activity against all phages, indicating that the predicted alpha-helical structure of this region is important. Mutations in an adjacent region of basic amino acids had various effects on phage resistance, suggesting that these basic residues are also important for AbiA activity. This directed mutagenesis analysis of AbiA indicated that the leucine repeat structure is essential for conferring phage resistance against three species of lactococcal bacteriophages.

Amino Acid Sequence↗
PubMed1998

Control of expression of LlaI restriction in Lactococcus lactis.

The plasmid encoded LlaI R/M system from Lactococcus lactis ssp. lactis consists of a bidomain methylase, with close evolutionary ties to type IIS methylases, and a trisubunit restriction complex. Both the methylase and restriction subunits are encoded on a polycistronic 6.9 kb operon. In this study, the 5' end of the llal 6.9 kb transcript was determined by primer extension analysis to be 254 bp upstream from the first R/M gene on the operon, llalM. Deletion of this promoter region abolished LlaI restriction in L. lactis. Analysis of the intervening sequence revealed a 72-amino-acid open reading frame, designated llalC, with a conserved ribosome binding site and helix-turn-helix domain. Overexpression of llalC in Escherichia coli with a T7 expression vector produced the predicted protein of 8.2 kDa. Mutation and in trans complementation analyses indicated that C-LlaI positively enhanced LlaI restriction activity in vivo. Northern analysis and transcriptional fusions of the llal promoter to a lacZ reporter gene indicated that C x LlaI did not enhance transcription of the llal operon. Databank searches with the deduced protein sequence for llalC revealed significant homologies to the E. coli Rop regulatory and mRNA stabilizer protein. Investigation of the effect of C x LlaI on enhancement of LlaI restriction in L. lactis revealed that growth at elevated temperatures (40 degrees C) completely abolished any enhancement of restriction activity. These data provide molecular evidence for a mechanism on how the expression of a restriction system in a prokaryote can be drastically reduced during elevated growth temperatures, by a small regulatory protein.

Amino Acid Sequence↗
PubMed1998

Molecular characterization of the Lactococcus lactis LlaKR2I restriction-modification system and effect of an IS982 element positioned between the restriction and modification genes.

The nucleotide sequence of the plasmid-encoded LlaKR2I restriction-modification (R-M) system of Lactococcus lactis subsp. lactis biovar diacetylactis KR2 was determined. This R-M system comprises divergently transcribed endonuclease (llaKR2IR) and methyltransferase (llaKR2IM) genes; located in the intergenic region is a copy of the insertion element IS982, whose putative transposase gene is codirectionally transcribed with llaKR2IM. The deduced sequence of the LlaKR2I endonuclease shared homology with the type II endonuclease Sau3AI and with the MutH mismatch repair protein, both of which recognize and cleave the sequence 5' GATC 3'. In addition, M. LlaKR2I displayed homology with the 5-methylcytosine methyltransferase family of proteins, exhibiting greatest identity with M. Sau3AI. Both of these proteins shared notable homology throughout their putative target recognition domains. Furthermore, subclones of the native parental lactococcal plasmid pKR223, which encode M. LlaKR2I, all remained undigested after treatment with Sau3AI despite the presence of multiple 5' GATC 3' sites. The combination of these data suggested that the specificity of the LlaKR2I R-M system was likely to be 5' GATC 3', with the cytosine residue being modified to 5-methylcytosine. The IS982 element located within the LlaKR2I R-M system contained at its extremities two 16-bp perfect inverted repeats flanked by two 7-bp direct repeats. A perfect extended promoter consensus, which represented the likely original promoter of the llaKR2IR gene, was shown to overlap the direct repeat sequence on the other side of IS982. Specific deletion of IS982 and one of these direct repeats via a PCR strategy indicated that the LlaKR2I R-M determinants do not rely on elements within IS982 for expression and that the efficiency of bacteriophage restriction was not impaired.

Amino Acid Sequence↗
PubMed1997

Evaluation of using a short region of the recA gene for rapid and sensitive speciation of dominant bifidobacteria in the human large intestine.

The feasibility of intragenerically characterizing bifidobacteria by a comparison of a short region within the recA gene was tested. An approximately 300 bp fragment of the recA gene was PCR-amplified from six species from the genus Bifidobacterium using primers directed to two universally conserved regions of the recA gene. A phylogenetic analysis of the sequenced recA products compared favorably to classification based on the 16S rRNA sequences of the species tested. To apply this rapid methodology to unknown human intestinal bifidobacteria, 46 isolates were randomly chosen from the feces of four subjects and initially characterized by RFLP analysis of a PCR-amplified region of their 16S RNA genes. From a representative of the dominant RFLP family in each of the subjects, the recA segment was PCR-amplified, sequenced and phylogenetically analyzed. All four isolates were found to be related to one another and to B. longum and B. infantis. These results illustrate that the recA gene may be useful for intrageneric phylogenetic analysis as well as for the identification of unknown fecal bifidobacteria.

Adolescent↗
PubMed1997

Differentiation of ingested and endogenous bifidobacteria by DNA fingerprinting demonstrates the survival of an unmodified strain in the gastrointestinal tract of humans.

Consumption of bifidobacteria as a dietary adjunct has received considerable attention for its possible role in the maintenance of gastrointestinal health. However, speculation exists about these presumed health benefits because of an inability to assess the fate and mechanism of action of ingested bifidobacteria. Thus, our objective was to examine the fate of ingested bifidobacteria through the gastrointestinal tract. Variations in the highly conserved 16S ribosomal DNA (rDNA) of bifidobacteria from six male subjects (18 to 35 y old) were assessed by restriction fragment length polymorphism (RFLP) analysis. During the 16-d study, 10(10) colony-forming units (CFU) of a commercially available bifidobacteria were delivered to subjects in fluid milk for each of 8 d. During the remaining 8 d, subjects consumed milk without bifidobacteria. Feces were collected at 4-d intervals and plated on selective media. For each subject, 10-15 colonies were randomly selected and used as template for PCR-amplification of 16S rDNA. 16S rDNA was restriction digested and resolved by electrophoresis. The 16S rDNA-RFLP of the ingested bifidobacteria was unique compared with bifidobacteria found in subjects prior to the feeding study. When subjects consumed bifidobacteria, a 16S rDNA-RFLP identical to that of the ingested bifidobacteria was observed in feces. The concentration of the ingested bifidobacteria in feces increased to 67.2 +/- 8.5% (mean +/- SEM) of total bifidobacteria. After feeding stopped, the ingested bifidobacteria diminished and became undetectable. Using this molecular approach to monitor ingested bifidobacteria, we demonstrate the kinetics of passage of this organism through the gastrointestinal tract of healthy humans.

Administration, Oral↗
PubMed1997

A triggered-suicide system designed as a defense against bacteriophages.

A novel bacteriophage protection system for Lactococcus lactis based on a genetic trap, in which a strictly phage-inducible promoter isolated from the lytic phage phi31 is used to activate a bacterial suicide system after infection, was developed. The lethal gene of the suicide system consists of the three-gene restriction cassette LlaIR+, which is lethal across a wide range of gram-positive bacteria. The phage-inducible trigger promoter (phi31P) and the LlaIR+ restriction cassette were cloned in Escherichia coli on a high-copy-number replicon to generate pTRK414H. Restriction activity was not apparent in E. coli or L. lactis prior to phage infection. In phage challenges of L. lactis(pTRK414H) with phi31, the efficiency of plaquing was lowered to 10(-4) and accompanied by a fourfold reduction in burst size. Center-of-infection assays revealed that only 15% of infected cells released progeny phage. In addition to phage phi31, the phi31P/LlaIR+ suicide cassette also inhibited four phi31-derived recombinant phages at levels at least 10-fold greater than that of phi31. The phi31P/LlaIR+-based suicide system is a genetically engineered form of abortive infection that traps and eliminates phages potentially evolving in fermentation environments by destroying the phage genome and killing the propagation host. This type of phage-triggered suicide system could be designed for any bacterium-phage combination, given a universal lethal gene and an inducible promoter which is triggered by the infecting bacteriophage.

Amino Acid Sequence↗
PubMed1996

Medial arterial calcification, calcific aortic stenosis and mitral annular calcification in a diabetic patient with severe autonomic neuropathy.

Medial arterial calcification (Mönckeberg's arteriosclerosis) is well described in diabetic patients with autonomic neuropathy. There is also a high prevalence of diabetes mellitus among subjects with calcific aortic stenosis and mitral annular calcification. We describe a diabetic patient with autonomic neuropathy and extensive medial arterial calcification who also had calcification of the aortic valve and of the mitral valve annulus. We propose that autonomic neuropathy may play a role in calcification of these structures at the base of the heart.

Adult↗
PubMed1996

Development of an expression strategy using a lytic phage to trigger explosive plasmid amplification and gene expression.

A novel plasmid-based expression strategy, exploiting two features of lytic bacteriophages, was developed in Lactococcus lactis. Components of this system include a phage origin of replication and phage expression signals, which were induced to high efficiency upon phage infection of the host. Phage-specific expression signals were cloned from phi 31 in a promoter-screening strategy using the lacZ gene from Streptococcus thermophilus. One clone exhibited a significant induction in beta-galactosidase production and concomitant increase in lacZ mRNA during the phi 31 infection cycle of the host. Molecular characterization of the cloned insert revealed 888 bp positioned near the phi 31 cos site. Primer extension analysis showed that transcription was induced approximately 20 min following phi 31 infection at four points, apparently organized in two sets of tandem promoters on the cloned phage insert. One of these middle phage promoters also showed a basal level of activity prior to phage infection. The phi 31 promoter lacZ cassette was cloned into a low-copy-number vector plasmid containing the phi 31 origin of replication (ori31) and the resulting low-copy-number plasmid exhibited negligible beta-galactosidase production in L. lactis. However, > 2,000 units were detected following a deliberate infection with phi 31. A control expression plasmid without ori31 could only be induced to 85 units. The combination of these phage-inducible expression signals together with ori31 functioned synergistically to drive rapid and high efficiency expression of a heterologous gene in L. lactis.

Amino Acid Sequence↗
PubMed1995

Iron-responsive gene expression in Pseudomonas fluorescens M114: cloning and characterization of a transcription-activating factor, PbrA.

In response to iron limitation. Pseudomonas fluorescens M114 induces a number of genes including an iron-scavenging siderophore termed pseudobactin M114, its cognate receptor, PbuA, and a casein protease. A Tn5lacZ-induced mutant (M114FA1) was isolated that exhibits a pleiotropic phenotype and lacks the ability to express these iron-regulated genes. A cosmid clone was identified which complements this mutation. This clone is capable of activating a number of iron-regulated promoter fusion constructs from P. fluorescens M114 and Pseudomonas putida WCS358 and can also promote expression of these fusions in Escherichia coli. A series of insertion mutants was constructed by homologous recombination which were unable to transcribe the promoter fusions. DNA sequence analysis of the complementing region identified one open reading frame (ORF) termed pbrA (pseudobactin regulation activation) and the deduced amino acid sequence shows domains with significant homology to a number of ECF (extracytoplasmic function) transcriptional regulators of the sigma 70 sigma factor family, including fecl required for expression of the ferric dicitrate outer-membrane receptor protein of E. coli. Sequences upstream of the pbrA gene suggest that transcription of pbrA may also be iron regulated.

Amino Acid Sequence↗
PubMed1995

Age at onset: the major determinant of outcome in Parkinson's disease.

Factors at presentation which influenced the course of the disease and response to treatment were assessed in 125 de novo patients with Parkinson's disease. Ninety-eight patients were available for re-assessment at 5 years. Older patients presented earlier after the onset of symptoms, deteriorated more rapidly, and were significantly more likely to develop dementia and impairment of balance. Increasing age and symmetrical disease predicted the new appearance of imbalance. Age of onset did not predict dyskinesia or end of dose failure. A low tremor score at baseline and female gender were predictive of the early appearance of dyskinesia. Patients who experienced end of dose failure were taking a significantly higher dose of levodopa. Once dose and duration of treatment were corrected for, no baseline features were predictive of end of dose failure. The dose of levodopa at 5 years was positively correlated to baseline disease severity as measured by the Columbia score. We conclude that the age of onset of symptoms of Parkinson's disease is a major determinant of the course of the disease and response to treatment.

Adult↗
PubMed1995

In vivo restriction by LlaI is encoded by three genes, arranged in an operon with llaIM, on the conjugative Lactococcus plasmid pTR2030.

The LlaI restriction and modification (R/M) system is encoded on pTR2030, a 46.2-kb conjugative plasmid from Lactococcus lactis. The llaI methylase gene, sequenced previously, encodes a functional type IIS methylase and is located approximately 5 kb upstream from the abiA gene, encoding abortive phage resistance. In this study, the sequence of the region between llaIM and abiA was determined and revealed four consecutive open reading frames (ORFs). Northern (RNA) analysis showed that the four ORFs were part of a 7-kb operon with llaIM and the downstream abiA gene on a separate transcriptional unit. The deduced protein sequence of ORF2 revealed a P-loop consensus motif for ATP/GTP-binding sites and a three-part consensus motif for GTP-binding proteins. Data bank searches with the deduced protein sequences for all four ORFs revealed no homology except for ORF2 with MerB, in three regions that coincided with the GTP-binding motifs in both proteins. To phenotypically analyze the llaI operon, a 9.0-kb fragment was cloned into a high-copy-number lactococcal shuttle vector, pTRKH2. The resulting construct, pTRK370, exhibited a significantly higher level of in vivo restriction and modification in L. lactis NCK203 than the low-copy-number parental plasmid, pTR2030. A combination of deletion constructions and frameshift mutations indicated that the first three ORFs were involved in LlaI restriction, and they were therefore designated llaI.1, llaI.2, and llaI.3. Mutating llaI.1 completely abolished restriction, while disrupting llaI.2 or llaI.3 allowed an inefficient restriction of phage DNA to occur, manifested primarily by a variable plaque phenotype. ORF4 had no discernible effect on in vivo restriction. A frameshift mutation in llaIM proved lethal to L. lactis NCK203, implying that the restriction component was active without the modification subunit. These results suggested that the LlaI R/M system is unlike any other R/M system studied to date and has diverged from the type IIS class of restriction enzymes by acquiring some characteristics reminiscent of type I enzymes.

Amino Acid Sequence↗
PubMed1994

Isolation of a gene (pbsC) required for siderophore biosynthesis in fluorescent Pseudomonas sp. strain M114.

An iron-regulated gene, pbsC, required for siderophore production in fluorescent Pseudomonas sp. strain M114 has been identified. A kanamycin-resistance cassette was inserted at specific restriction sites within a 7 kb genomic fragment of M114 DNA and by marker exchange two siderophore-negative mutants, designated M1 and M2, were isolated. The nucleotide sequence of approximately 4 kb of the region flanking the insertion sites was determined and a large open reading frame (ORF) extending for 2409 bp was identified. This gene was designated pbsC (pseudobactin synthesis C) and its putative protein product termed PbsC. PbsC was found to be homologous to a family of enzymes involved in the biosynthesis of secondary metabolites, including EntF of Escherichia coli. These enzymes are believed to act via ATP-dependent binding of AMP to their substrate. Several areas of high sequence homology between these proteins and PbsC were observed, including a conserved AMP-binding domain. The expression of pbsC is iron-regulated as revealed when a DNA fragment containing the upstream region was cloned in a promoter probe vector and conjugated into the wild-type strain, M114. The nucleotide sequence upstream of the putative translational start site contains a region homologous to previously defined -16 to -25 sequences of iron-regulated genes but did not contain an iron-box consensus sequence. It was noted that inactivation of the pbsC gene also affected other iron-regulated phenotypes of Pseudomonas M114.

Amino Acid Sequence↗
PubMed1994

Escherichia coli ferric uptake regulator (Fur) can mediate regulation of a pseudomonad iron-regulated promoter.

Introduction of a Pseudomonas iron-regulated promoter lacZ fusion (SP1) and a Pseudomonas transcriptional factor into Escherichia coli allowed expression of the promoter in this heterologous host. Evaluation of this promoter in wild-type and fur mutants of E. coli, by measuring beta-galactosidase activity, indicated that E. coli Fur can regulate the Pseudomonas promoter in response to iron starvation. Gel retardation assays suggested that purified Fur protein could interact with the SP1 promoter upstream of the transcriptional start. DNase I footprinting analysis established that Fur protected a primary 58-bp region (-50 to -106 bp). These protein/DNA interactions correlate with the observed in vivo regulation of the SP1 promoter in E. coli and indicate that Fur can functionally regulate a Pseudomonas iron-regulated promoter.

Bacterial Proteins↗
PubMed1994

The Sydney Multicentre Study of Parkinson's disease: a randomised, prospective five year study comparing low dose bromocriptine with low dose levodopa-carbidopa.

149 previously untreated patients with Parkinson's disease were recruited over a three year period and randomly allocated to either low dose levodopa-carbidopa (< or = 600/150 mg/day) or low dose bromocriptine (< or = 30 mg/day). A five year follow up is reported on the 126 patients who completed the dose titration and who have not developed features of atypical Parkinsonism. Levodopa-carbidopa in low dosage adequately controlled symptoms in most patients and delayed the appearance of dyskinesia and end of dose failure for about two years longer than conventional doses. Only a few patients could be managed for more than one year on low dose bromocriptine alone; these patients had mild disease and asymmetric signs. Patients randomised to bromocriptine did not develop dyskinesia or troublesome end of dose failure until levodopa-carbidopa was added. The prevalence of dyskinesia in this group was lower than in patients given levodopa-carbidopa alone. The prevalence of end of dose failure was similar in the two randomisation groups once levodopa was introduced.

Adult↗