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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
PubMed2001

Lesion of thalamic centromedian--parafascicular complex after chronic deep brain stimulation.

A patient with PD who exhibited disabling tremor and prominent dyskinesia underwent deep brain stimulation (DBS) of the left thalamic ventral intermediate nucleus. The electrode migrated and was replaced but with suboptimal clinical response. Two years later, postmortem analysis found the second electrode tip had entered the thalamic centromedian-parafascicular complex. There was a small thalamotomy and cell loss exceeding that found in PD. Thalamic damage may occur in association with DBS for PD.

Chronic Disease↗
PubMed2001

Screening of intestinal microflora for effective probiotic bacteria.

Increasing consumer awareness of health-promoting intestinal bacteria has fueled the addition of viable probiotic bacteria as functional ingredients in certain foods. However, to effectively market the enhanced attributes of these foods, the added probiotic bacteria need to have scientific credibility. The scientific rationale for using many of the strains of probiotic bacteria currently on the market is weak. Furthering the current understanding of what features a bacterium needs to have for effective probiotic functionality will enable the selection of strains with a more credible scientific rationale. To screen for effective strains, one must understand the microbial diversity in the intestines of healthy individuals. The advent of molecular tools has greatly enhanced our ability to accomplish this. These tools comprise genetic fingerprinting, specific probes, molecular speciation, and techniques for the in situ analysis of specific microbial groups in the intestine. This review will detail these scientific approaches and how their impact will improve criteria for selection of probiotic bacteria.

Bifidobacterium↗
PubMed2001

Isolation, characterization, and influence of native, nonstarter lactic acid bacteria on Cheddar cheese quality.

To determine whether adventitious nonstarter lactic acid bacteria (NSLAB) might affect cheese flavor and quality, we studied a population of NSLAB present in 30 premium quality Cheddar cheeses (3-mo ripened) produced at a commercial facility in the United States. DNA fingerprinting analysis with a sensitive strategy for arbitrary priming polymerase chain reaction showed that 75 isolates corresponded to at least 18 distinct nonstarter organisms. According to ribotype database comparisons of representatives from the 18 groups, 9 matched Lactobacillus (closest to paracasei species), 8 matched Streptococcus thermophilus, and 1 matched to a Lactococcus species. This finding indicated that among the 75 NSLAB isolates, Lactobacillus made up 64%, S. thermophilus 32%, and Lactococcus 4%. Isolates representing 11 NSLAB groups were characterized for protease, peptidase, and diacetyl production. Based on this phenotypic analysis, two Lactobacillus isolates were evaluated as adjuncts in Cheddar cheese. All of the NSLAB identified from the adjunct cheese at 3 mo by DNA fingerprinting consisted of the adjunct lactobacilli, showing that the adjunct strains predominated throughout the early stages of ripening. The impact of adjunct lactobacilli was evident after 6 mo when free amino acids significantly increased and sensory scores improved in adjunct cheese as compared with a control cheese. The largest impact was found in adjunct cheese containing a blend of both lactobacilli strains. These results show that certain adventitious NSLAB positively contribute to flavor development.

Amino Acids↗
PubMed2000

Use of a single, triplicate arbitrarily primed-PCR procedure for molecular fingerprinting of lactic acid bacteria.

Arbitrarily primed (AP)-PCR can be used to generate characteristic DNA fingerprint patterns. However, small changes in reaction conditions can cause band irreproducibility. In this study, a single methodology encompassing triplicate reactions, which were intentionally exposed to three different annealing temperatures, enabled bands that were reproducibly generated to be recognized. A single triplicate AP-PCR (TAP-PCR) procedure, using an 18-mer primer, was developed and used to fingerprint representative isolates from the major genera of lactic acid bacteria and Bifidobacterium to the strain level.

Bacteria↗
PubMed2000

Characterization of AbiR, a novel multicomponent abortive infection mechanism encoded by plasmid pKR223 of Lactococcus lactis subsp. lactis KR2.

The native lactococcal plasmid pKR223 encodes two distinct phage resistance mechanisms, a restriction and modification (R/M) system designated LlaKR2I and an abortive infection mechanism (Abi) which affects prolate-headed-phage proliferation. The nucleotide sequence of a 16,174-bp segment of pKR223 encompassing both the R/M and Abi determinants has been determined, and sequence analysis has validated the novelty of the Abi system, which has now been designated AbiR. Analysis of deletion and insertion clones demonstrated that AbiR was encoded by two genetic loci, separated by the LlaKR2I R/M genes. Mechanistic studies on the AbiR phenotype indicated that it was heat sensitive and that it impeded phage DNA replication. These data indicated that AbiR is a novel multicomponent, heat-sensitive, "early"-functioning Abi system and is the first lactococcal Abi system described which is encoded by two separated genetic loci.

Bacteriophages↗
PubMed2000

Use of chemical mutagenesis for the isolation of food grade beta-galactosidase overproducing mutants of bifidobacteria, lactobacilli and Streptococcus thermophilus.

A classical chemical mutagenesis protocol was evaluated for increasing beta-galactosidase production by probiotic bacteria to improve their potential to treat symptoms of lactose malabsorption in humans. Two Bifidobacterium species (B. breve and B. longum) and one strain each of Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus thermophilus were tested by a single exposure to two chemical mutagens, ethyl methanesulfonate (EMS) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). To screen for beta-galactosidase (beta-gal) overproducing mutants, optimized EMS and MNNG mutant pots for each strain were plated on BHI agar containing 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-gal). Colonies that exhibited a blue color were selected for quantitative beta-gal activities using the o-nitrophenyl-beta-galactoside (ONPG) assay. Seventy-five mutants were obtained out of more than 2 million colonies screened and showed increased beta-galactosidase activities compared with the wild-type strains. EMS gave a higher frequency of beta-gal overproducing mutants than MNNG for three of the four strains, S. thermophilus, B. breve, and B. longum, whereas the frequency of L. delbrueckii ssp. bulgaricus beta-gal mutants was similar with both mutagens. The highest beta-gal increases, when induced during growth in lactose, for mutants of each culture were 137% for L. delbrueckii ssp. bulgaricus; 104% for S. thermophilus; 70% for B. breve; and 222% for B. longum mutants. This food-grade classical approach has the ability to moderately increase beta-gal concentrations in probiotic cultures to improve their potential for treating the symptoms of lactose malabsorption in humans.

Bifidobacterium↗
PubMed2000

Identification of a gene cluster encoding Krebs cycle oxidative enzymes linked to the pyruvate carboxylase gene in Lactococcus lactis ssp. lactis C2.

We identified a 14-kb pyruvate carboxylase gene-containing fragment from a lactococcal C2-lambda phage genomic library. Downstream of the pyruvate carboxylase gene-containing fragment, a gene cluster coding for open reading frames displaying extensive homology to citrate synthase, aconitase, and a truncated isocitrate dehydrogenase was identified. However, the truncation was shown to have occurred during the cloning by two noncontiguous Sau3AI fragments ligating together. The lactococcal citrate synthase gene consisted of 1323 bp and encoded a 441-amino acid citrate synthase protein. The lactococcal aconitase gene was 2544 bp and encoded an 848-amino acid protein. Corresponding to the complete citrate synthase gene, citrate synthase activity was detected in Lactococcus lactis ssp. lactis C2. Isocitrate dehydrogenase activity was found to be missing in Lactococcus lactis C2, suggesting that the gene may be incomplete or is not expressed, resulting in a requirement for glutamic acid in lactococci.

Aconitate Hydratase↗
PubMed2000

Classification of a bacterial isolate, from pozol, exhibiting antimicrobial activity against several gram-positive and gram-negative bacteria, yeasts, and molds.

A bacterial isolate, designated CS93, capable of producing a broad-spectrum antimicrobial compound(s) effective against gram-positive and gram-negative bacteria, yeasts, and molds was isolated from pozol, a fermented maize product. This strain was phenotypically similar to another pozol isolate that was previously designated as Agrobacterium azotophilium by other investigators. By using biochemical, phenotypic, and 16S rRNA sequence analysis, both pozol isolates were identified as members of the genus Bacillus, possibly a variant of Bacillus subtilis. While the antimicrobial compound(s) was initially produced only on a solid medium, parameters were identified for production in broth. The compound(s) was heat stable (121 degrees C for 15 min), exhibited activity over a wide pH range (pH 3 to pH 11), and was inactivated by pronase E. The antimicrobial compound(s) was bactericidal and bacteriolytic against Escherichia coli V517, bacteriostatic against Micrococcus luteus, and fungistatic against Saccharomyces cerevisiae. The inhibitory compound(s) could possibly serve as a food biopreservative.

Animals↗
PubMed2000

Methods for analysis of the intestinal microflora.

The concept of probiotics has been around for about 100 years. Yet its impact on human nutrition is still an emerging concept. Lack of convincing scientific validation for the efficacy of any ingested probiotic bacterium on intestinal health, has been a major reason for the low impact of probiotics on human nutrition. Obtaining positive scientific validation requires the use of suitable probiotic strains and also the necessary tools to monitor the performance of these bacteria in the intestines of individuals. To date, selection of strains for probiotic purposes has not been based on a scientific directed approach, primarily because it is not yet fully known what specific traits a desirable probiotic strain should possess. Filling this knowledge void will depend largely on furthering our understanding of the human intestinal ecosystem and the functional role of specific bacteria for intestinal health. Traditional approaches for studying this ecosystem have provided a good foundation in this knowledge base. Complementation of the traditional approaches with the emergence of sophisticated molecular tools shows enormous promise for obtaining the necessary insight into the intestinal microflora. This review will cover the traditional methodologies which have been used to analyze the human intestinal microflora. It will also reveal the development of modern molecular approaches for studying the diversity and phylogeny of its flora, and the rapid molecular tools for monitoring the presence of specific strains in the intestine. Finally, it will address the advent of in situ analysis of individual microbial cells, which promises to provide tremendous advances in our understanding of the microflora and their metabolic activities in the human intestine.

Bacteria↗
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↗