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Biomedical subjects

J Kok

Publications and source records attributed to J Kok.

At least 19 recordsLinked to original sources
PubMed2006

Immunogenicity of a malaria parasite antigen displayed by Lactococcus lactis in oral immunisations.

A putative protective protein from Plasmodium falciparum merozoites, MSA2, was expressed in two different ways on the cell surface of the Gram-positive food-grade bacterium, Lactococcus lactis. The first display format exploits an LPXTG-type anchoring motif of the lactococcal proteinase PrtP to covalently anchor MSA2 to the genetically modified producer cells. In a second display format, MSA2 was fused to the peptidoglycan-binding domain (Protein Anchor) of the lactococcal cell wall hydrolase AcmA and was non-covalently rebound to the surface of non-genetically modified, non-living high-binder L. lactis cells, termed Gram-positive enhancer matrix (GEM) particles. The L. lactis recombinants carrying covalently bound MSA2 were used to immunise rabbits through nasal and oral routes. The highest levels of IgG antibodies reacting with near-native MSA2 on merozoites was elicited by oral administration. Intestinal antibodies to MSA2 were produced only after oral immunisation. MSA2-specific T(h)-cell activation could be demonstrated. Based on these results, the immunogenicity in oral immunisations of MSA2, bound non-covalently to non-genetically modified L. lactis GEM particles, was compared with MSA2 that was bound covalently to genetically modified L. lactis. These two forms elicited similar titres of serum antibodies. The results illustrate the potential of using non-genetically modified L. lactis as a safe vaccine delivery vehicle to elicit systemic antibodies, thereby avoiding the dissemination of recombinant DNA into the environment.

Administration, Intranasal↗
PubMed2004

Antenatal use of a novel vaginal birth training device by term primiparous women in Singapore.

INTRODUCTION: To study the use, safety and effectiveness of a novel antenatal vaginal birth training device (EPI-NO) in primiparous women. METHODS: Antenatal use of the EPI-NO vaginal birth trainer was prospectively studied in 31 primiparous booked patients who were delivered by obstetricians from July to December 2002 at the KK Women's and Children's Hospital. This was studied in relation to episiotomy rate, perineal trauma and analgesic requirements during the postpartum period. A patient questionnaire form was used to assess their perception of pain and how well they coped with its use. For comparison, perineal trauma was also studied retrospectively in 60 consecutive obstetrician-booked primiparous term patients who had normal vaginal delivery (NVD) and who did not use EPI-NO during the same study period. RESULTS: The mean length of usage was for 2.1 weeks (standard deviation [sd] 1.2 weeks). The mean frequency of use was 5.3 episodes per week (sd 2.1, range 1 to 7). There was no laceration and vaginal infection arising from its usage. There was a case of minimal bleeding post-usage. There were 20 (64.5 percent) NVDs, four (12.9 percent) forceps deliveries, five vacuum deliveries (16.1 percent) and two (6.5 percent) Caesarean sections. Of the 29 vaginal delivery cases, 19 (65.5 percent) had episiotomy, eight (27.6 percent) had lacerations, and two (6.9 percent) did not sustain laceration. The reasons for episiotomy in the 19 cases were nine cases of pending tearing of vagina/perineum, nine cases of instrumental vaginal deliveries, and one to shorten second stage. There was no third degree tear. 21 (67.7 percent) out of 30 required a painkiller. The majority of patients (17; 54.8 percent) appeared to be comfortable with the use of EPI-NO. All coped well with vaginal examination after using EPI-NO perineal training. Comparing among term primiparous NVD cases with (n value equals 20) and without (n value equals 60) EPI-NO, the perineal trauma rate (90.0 percent vs 96.6 percent, p value equals 0.24) was slightly but not significantly lower in the EPI-NO group. The episiotomy rate was significantly lower (50.0 percent vs 93.3 percent, p value is less than 0.0001) and the extent of perineal trauma in the patient appeared to be less severe in cases using EPI-NO. CONCLUSION: EPI-NO appeared to be safe and acceptable to the majority of users. Although birth training with EPI-NO significantly decreases the rate of episiotomies in term primiparous patients, and the degree of perineal tissue injury appeared to be less in the EPI-NO group especially among those with lacerations, the overall perineal trauma rate was slightly but not significantly lower, in view of the higher spontaneous laceration rate in the EPI-NO group.

Analgesics↗
PubMed2002

Reflective journal writing: how it promotes reflective thinking in clinical nursing education: a students' perspective.

According to the outcomes-based education and training system of education (OBET) in the country and the South African Nursing Council, education should focus on "creating" reflective learners and practitioners. This article seeks to determine the effectiveness of reflective journal writing in promoting the reflective thinking of learners in clinical nursing education and to validate the guidelines described in a bigger study on how to facilitate reflective thinking using reflective journal writing. A qualitative, contextual, explorative, descriptive research design was used to determine the learners' perceptions on whether reflective journal writing did promote their higher-level thinking skills during the six-month placement in a psychiatric clinical practice using the reflective diaries. From a population of seventeen fourth-year students, six volunteered to participate in a focus group interview. The data was analysed by means of the descriptive method of open coding of Tesch (in Creswell, 1994:154-156). Positive and negative results from the perceptions of the participants and a literature review served as a basis for deducing and describing guidelines for the effective use of reflective journal writing in promoting reflective thinking in clinical nursing education. The positive perception was the development of problem-solving skills attained through reflection by using analytical critical thinking, synthesis and the evaluation of situations. Self-evaluation leading to intellectual growth and self-awareness indicated a positive perception. Negative perceptions were that reflective journal writing is time consuming, content based with a lack of clear expectations from the teacher, and distrust of students about the information written. Guba's model of ensuring trustworthiness in qualitative research as described in Krefting (1991:215-222) was employed. It is concluded that reflective journal writing in clinical nursing education does promote reflective thinking and learning. The use of the developed guidelines will minimise the negative perceptions. It is recommended that the guidelines be used in basic training at all levels of training to attain the objective of nursing education and training, which is to produce a critical, analytical thinker through reflection to improve practice.

Attitude of Health Personnel↗
PubMed2001

Proteins of the lactococcin A secretion system: lcnD encodes two in-frame proteins.

Polyclonal antibodies were raised against LcnC and LcnD proteins of the Lactococcus lactis bacteriocin lactococcin A secretory system to examine their cellular location and interaction. Two major reacting bands were detected by Western immunoblot with the anti-LcnD antibody: one of 52 kDa (LcnD) and another of 45 kDa, called here LcnD*. LcnD* was still detectable after removing the AUG start codon for LcnD. Chemical cross-linking analyses of membrane fractions of L. lactis cells expressing the LcnC/D secretion machinery were performed. Our results indicate that LcnD is present in the secretion machinery complex as a dimer and is able to interact with LcnD* and LcnC.

ATP-Binding Cassette Transporters↗
PubMed2000

The anaerobic (class III) ribonucleotide reductase from Lactococcus lactis. Catalytic properties and allosteric regulation of the pure enzyme system.

Lactococcus lactis contains an operon with the genes (nrdD and nrdG) for a class III ribonucleotide reductase. Strict anaerobic growth depends on the activity of these genes. Both were sequenced, cloned, and overproduced in Escherichia coli. The corresponding proteins, NrdD and NrdG, were purified close to homogeneity. The amino acid sequences of NrdD (747 residues, 84.1 kDa) and NrdG (199 residues, 23.3 kDa) are 53 and 42% identical with the respective E. coli proteins. Together, they catalyze the reduction of ribonucleoside triphosphates to the corresponding deoxyribonucleotides in the presence of S-adenosylmethionine, reduced flavodoxin or reduced deazaflavin, potassium ions, dithiothreitol, and formate. EPR experiments demonstrated a [4Fe-4S](+) cluster in reduced NrdG and a glycyl radical in activated NrdD, similar to the E. coli NrdD and NrdG proteins. Different from E. coli, the two polypeptides of NrdD and the proteins in the NrdD-NrdG complex were only loosely associated. Also the FeS cluster was easily lost from NrdG. The substrate specificity and overall activity of the L. lactis enzyme was regulated according to the general rules for ribonucleotide reductases. Allosteric effectors bound to two separate sites on NrdD, one binding dATP, dGTP, and dTTP and the other binding dATP and ATP. The two sites showed an unusually high degree of cooperativity with complex interactions between effectors and a fine-tuning of their physiological effects. The results with the L. lactis class III reductase further support the concept of a common origin for all present day ribonucleotide reductases.

Allosteric Regulation↗
PubMed2000

Current strategies for improving food bacteria.

Novel concepts and methodologies are emerging that hold great promise for the directed improvement of food-related bacteria, specifically lactic acid bacteria. Also, the battle against food spoilage and pathogenic bacteria can now be fought more effectively. Here we describe recent advances in microbial physiology and genomic research of these organisms that enable novel strategies for obtaining safe, healthy, and good-tasting fermented food products.

Bacteriocins↗
PubMed2000

Lactic acid bacteria: the bugs of the new millennium.

Lactic acid bacteria (LABs) are widely used in the manufacturing of fermented food and are among the best-studied microorganisms. Detailed knowledge of a number of physiological traits has opened new potential applications for these organisms in the food industry, while other traits might be beneficial for human health. Important new developments have been made in the research of LABs in the areas of multidrug resistance, bacteriocins and quorum sensing, osmoregulation, proteolysis, autolysins and bacteriophages. Recently, progress has been made in the construction of food-grade genetically modified LABs.

Bacteriocins↗
PubMed2000

Biological and molecular characterization of a two-peptide lantibiotic produced by Lactococcus lactis IFPL105.

The lactic acid bacterium Lactococcus lactis IFPL105 secretes a broad spectrum bacteriocin produced from the 46 kb plasmid pBAC105. The bacteriocin was purified to homogeneity by ionic and hydrophobic exchange and reverse-phase chromatography. Bacteriocin activity required the complementary action of two distinct peptides (alpha and beta) with average molecular masses of 3322 and 2848 Da, respectively. The genes encoding the two peptides were cloned and sequenced and were found to be identical to the ltnAB genes from plasmid pMRC01 of L. lactis DPC3147. LtnA and LtnB contain putative leader peptide sequences similar to the known 'double glycine' type. The predicted amino acid sequence of mature LtnA and LtnB differed from the amino acid content determined for the purified alpha and beta peptides in the residues serine, threonine, cysteine and alanine. Post-translational modification, and the formation of lanthionine or methyllanthionine rings, could partly explain the difference. Hybridization experiments showed that the organization of the gene cluster in pBAC105 responsible for the production of the bacteriocin is similar to that in pMRC01, which involves genes encoding modifying enzymes for lantibiotic biosynthesis and dual-function transporters. In both cases, the gene clusters are flanked by IS946 elements, suggesting an en bloc transposition. The findings from the isolation and molecular characterization of the bacteriocin provide evidence for the lantibiotic nature of the two peptides.

Amino Acid Sequence↗
PubMed2000

Broad-host-range shuttle vectors for screening of regulated promoter activity in viridans group streptococci: isolation of a pH-regulated promoter.

Viridans group streptococci are major constituents of the normal human oral flora and are also identified as the predominant pathogenic bacteria in native valve infective endocarditis. Little information is available regarding the regulation of gene expression in viridans group streptococci, either in response to changes in the oral environment or during development of endocarditis. We therefore constructed a set of broad-host-range vectors for the isolation of promoters from viridans group streptococci that are activated by specific environmental stimuli in vitro or in vivo. A genomic library of Streptococcus gordonii strain CH1 was constructed in one of the new vectors, and this library was introduced into a homologous bacterium by using an optimized electroporation protocol for viridans group streptococci. Because viridans group streptococci entering the bloodstream from the oral cavity encounter an increase in pH, we selected promoters upregulated by this specific stimulus. One of the selected promoter sequences showed homology to the promoter region of the hydA gene from Clostridium acetobutylicum, the expression of which is known to be regulated by the environmental pH. The isolation of this pH-regulated promoter shows that S. gordonii can sense an increase in the environmental pH, which serves as a signal for bacterial gene activation. Furthermore, this demonstrates the usefulness of these new selection vectors in research on adaptive gene expression of viridans group streptococci and possibly also of other gram-positive bacteria.

Base Sequence↗
PubMed2000

Bile salt hydrolase of Bifidobacterium longum-biochemical and genetic characterization.

A bile salt hydrolase (BSH) was isolated from Bifidobacterium longum SBT2928, purified, and characterized. Furthermore, we describe for the first time cloning and analysis of the gene encoding BSH (bsh) in a member of the genus Bifidobacterium. The enzyme has a native molecular weight of 125,000 to 130,000 and a subunit molecular weight of 35,024, as determined from the deduced amino acid sequence, indicating that the enzyme is a tetramer. The pH optimum of B. longum BSH is between 5 and 7, and the temperature optimum is 40 degrees C. The enzyme is strongly inhibited by thiol enzyme inhibitors, indicating that a Cys residue is likely to be involved in the catalytic reaction. The BSH of B. longum can hydrolyze all six major human bile salts and at least two animal bile salts. A slight preference for glycine-conjugated bile acids was detected based on both the specificity and the K(m) values. The nucleotide sequence of bsh was determined and used for homology studies, transcript analysis, and construction and analysis of various mutants. The levels of homology with BSH of other bacteria and with penicillin V acylase (PVA) of Bacillus sphaericus were high. On the basis of the similarity of BSH and PVA, whose crystal structure has been elucidated, BSH can be classified as an N-terminal nucleophile hydrolase with Cys as the N-terminal amino acid. This classification was confirmed by the fact that a Cys1Ala exchange by site-directed mutagenesis resulted in an inactive protein. Reverse transcription-PCR experiments revealed that bsh is part of an operon containing at least two genes, bsh and glnE (GlnE is glutamine synthetase adenylyltransferase). Two UV-induced BSH-negative mutants and one spontaneous BSH-negative mutant were isolated from B. longum SBT2928 cultures and characterized. These mutants had point mutations that inactivated bsh by premature termination, frameshift, or amino acid exchange.

Actinomycetales Infections↗
PubMed2000

Requirement of autolytic activity for bacteriocin-induced lysis.

The bacteriocin produced by Lactococcus lactis IFPL105 is bactericidal against several Lactococcus and Lactobacillus strains. Addition of the bacteriocin to exponential-growth-phase cells resulted in all cases in bacteriolysis. The bacteriolytic response of the strains was not related to differences in sensitivity to the bacteriocin and was strongly reduced in the presence of autolysin inhibitors (Co(2+) and sodium dodecyl sulfate). When L. lactis MG1363 and its derivative deficient in the production of the major autolysin AcmA (MG1363acmADelta1) were incubated with the bacteriocin, the latter did not lyse and no intracellular proteins were released into the medium. Incubation of cell wall fragments of L. lactis MG1363, or of L. lactis MG1363acmADelta1 to which extracellular AcmA was added, in the presence or absence of the bacteriocin had no effect on the speed of cell wall degradation. This result indicates that the bacteriocin does not degrade cell walls, nor does it directly activate the autolysin AcmA. The autolysin was also responsible for the observed lysis of L. lactis MG1363 cells during incubation with nisin or the mixture of lactococcins A, B, and M. The results presented here show that lysis of L. lactis after addition of the bacteriocins is caused by the resulting cell damage, which promotes uncontrolled degradation of the cell walls by AcmA.

Bacteriocins↗
PubMed2000

Heterologous coproduction of enterocin A and pediocin PA-1 by Lactococcus lactis: detection by specific peptide-directed antibodies.

Antibodies against enterocin A were obtained by immunization of rabbits with synthetic peptides PH4 and PH5 designed, respectively, on the N- and C-terminal amino acid sequences of enterocin A and conjugated to the carrier protein KLH. Anti-PH4-KLH antibodies not only recognized enterocin A but also pediocin PA-1, enterocin P, and sakacin A, three bacteriocins which share the N-terminal class IIa consensus motif (YGNGVXC) that is contained in the sequence of the peptide PH4. In contrast, anti-PH5-KLH antibodies only reacted with enterocin A because the amino acid sequences of the C-terminal parts of class IIa bacteriocins are highly variable. Enterocin A and/or pediocin PA-1 structural and immunity genes were introduced in Lactococcus lactis IL1403 to achieve (co)production of the bacteriocins. The level of production of the two bacteriocins was significantly lower than that obtained by the wild-type producers, a fact that suggests a low efficiency of transport and/or maturation of these bacteriocins by the chromosomally encoded bacteriocin translocation machinery of IL1403. Despite the low production levels, both bacteriocins could be specifically detected and quantified with the anti-PH5-KLH (anti-enterocin A) antibodies isolated in this study and the anti-PH2-KLH (anti-pediocin PA-1) antibodies previously generated (J. M. Martínez, M. I. Martínez, A. M. Suárez, C. Herranz, P. Casaus, L. M. Cintas, J. M. Rodríguez, and P. E. Hernández, Appl. Environ. Microbiol. 64:4536-4545, 1998). In this work, the availability of antibodies for the specific detection and quantification of enterocin A and pediocin PA-1 was crucial to demonstrate coproduction of both bacteriocins by L. lactis IL1403(pJM04), because indicator strains that are selectively inhibited by each bacteriocin are not available.

Animals↗
PubMed2000

The discrepancy between maturation of visual-evoked potentials and cognitive outcome at five years in very preterm infants with and without hemodynamic signs of fetal brain-sparing.

OBJECTIVE: After intrauterine growth restriction we found at the age of 6 months an acceleration of neurophysiologic maturation. However, at later ages impaired cognitive outcome has been reported. Therefore, we investigated in children with and without fetal hemodynamic adaptation to intrauterine growth restriction whether the accelerated neurophysiologic maturation in infancy might be associated with impaired cognitive outcome at preschool age. DESIGN: At 5 years of age cognitive function was assessed using the Revision of the Amsterdam Children's Intelligence Test in 73 preterm infants (26-33 weeks) who were prospectively followed from the antenatal period up to the age of 5 years. Maternal educational level was used as a background variable to estimate the confounding effects of socioeconomic status on cognitive function. Fetal Doppler studies were performed and the umbilical artery pulsatility index (PI) divided by the middle cerebral artery PI ratio (U/C ratio) was calculated. A U/C ratio >0.725 was considered as an indication of fetal cerebral hemodynamic adaptation to a compromised placental perfusion, ie, fetal brain-sparing. Visual-evoked potentials (VEPs) were recorded at 6 months and 1 year of age. In addition, data on neurologic status at 3 years were available. RESULTS: Mean IQ score was significantly lower for children born with a raised U/C ratio (87 +/- 16) compared with children with a normal U/C ratio (96 +/- 17). VEP latencies decreased significantly in infants with a normal U/C ratio, whereas no decrease was found in infants with a raised U/C ratio. Variables contributing significantly to the variance of cognitive function were: U/C group, VEP latency maturation, level of maternal education, and neurodevelopmental outcome at 3 years. The linear regression model explained 33% of the variance in cognitive function. CONCLUSIONS: Both being born with a raised U/C ratio and an acceleration of VEP latencies are negatively associated with cognitive outcome at 5 years of age. Fetal brain-sparing, although a seemingly beneficial adaptive mechanism for intact neurologic survival, is, however, later associated with a poorer cognitive outcome.

Adaptation, Physiological↗
PubMed1999

Membrane topology of the lactococcal bacteriocin ATP-binding cassette transporter protein LcnC. Involvement of LcnC in lactococcin a maturation.

Many non-lantibiotic bacteriocins of lactic acid bacteria are produced as precursors with N-terminal leader peptides different from those present in preproteins exported by the general sec-dependent (type II) secretion pathway. These bacteriocins utilize a dedicated (type I) secretion system for externalization. The secretion apparatus for the lactococcins A, B, and M/N (LcnA, B, and M/N) from Lactococcus lactis is composed of the two membrane proteins LcnC and LcnD. LcnC belongs to the ATP-binding cassette transporters, whereas LcnD is a protein with similarities to other accessory proteins of type I secretion systems. This paper shows that the N-terminal part of LcnC is involved in the processing of the precursor of LcnA. By making translational fusions of LcnC to the reporter proteins beta-galactosidase (LacZ) and alkaline phosphatase (PhoA*), it was shown that both the N- and C-terminal parts of LcnC are located in the cytoplasm. As the N terminus of LcnC is required for LcnA maturation and is localized in the cytoplasm, we conclude that the processing of the bacteriocin LcnA to its mature form takes place at the cytosolic side of the cytoplasmic membrane.

ATP-Binding Cassette Transporters↗
PubMed1999

Disgust sensitivity and the sex difference in fears to common indigenous animals.

Davey's mediational hypothesis [Davey, G. C. L. (1994). Self-reported fears to common indigenous animals in an adult UK population: the role of disgust sensitivity. British Journal of Psychology, 85, 541-554.] suggests that the sex difference in self-assessed animal fears can be accounted for by the sex difference in disgust sensitivity. An empirical test failed to support this hypothesis in a non-clinical sample (N = 214). Holding constant the influences of confounders such as age, fear of contamination, sex roles, neuroticism, psychoticism and disgust sensitivity, biological sex kept emerging as a significant predictor in relation to four types of animal fears (fear-relevant animals, dry or non-slimy invertebrates, slimy or wet looking animals and farm animals). Other things being equal, high disgust sensitivity either lost its predictive capability (in relation to dry or non-slimy invertebrates and slimy or wet looking animals) or predicted high fear of fear-relevant animals and of farm animals inequivalently across, respectively, the sexes (high in females only) and age groups (high in the old only). A multifactorial, interactionist approach should be advocated in the study of the aetiology of animal fears if progress in this area is to be achieved.

Adolescent↗
PubMed1999

Two trials of antenatal thyrotrophin-releasing hormone for fetal maturation: stopping before the due date. Antenatal TRH Trial and the Thyroneth Trial Groups.

OBJECTIVES: To determine whether the addition of antenatal thyrotrophin-releasing hormone (TRH) to corticosteroids, given to women at risk of preterm delivery, would decrease the risk of death and severe morbidity associated with prematurity. DESIGN: Two concurrent multicentre, double blind, randomised, placebo-controlled trials were designed with a common core dataset to be analysed as a single large pragmatic trial. SETTING: Hospital maternity units. POPULATIONS: Pregnant women at risk of preterm delivery who had been prescribed a course of corticosteroids to enhance fetal maturation. INTERVENTIONS: Antenatal administration of TRH 400 microg every eight hours for four doses versus matching placebo. MAIN OUTCOME MEASURES: Primary: death of the baby or chronic lung disease (defined as oxygen dependency at 28 days after birth). Secondary: other measures of respiratory morbidity, in particular respiratory distress syndrome. Other measures of short term neonatal morbidity including intraventricular haemorrhage and necrotising enterocolitis. Measures of maternal side effects. RESULTS: The antenatal TRH trial was halted early on the basis of external evidence. Overall a total of 225 women were recruited who delivered 275 babies. The primary outcome of death or chronic lung disease occurred in 33 babies in the TRH group and 43 babies in the placebo group (RR 0.8, 95% CI 0.5-1.2). There were no other differences between the two groups. Stratified analysis did not reveal any differences between the two groups depending on how long before the time of delivery the mother had received the TRH or placebo. CONCLUSIONS: These trials are too small to provide convincing evidence of the effect of antenatal TRH on neonatal outcome. When added to the existing systematic review and meta-analysis, however, these data should provide evidence on which subsequent practice can be based. The process by which the trials were monitored and stopped is of relevance to future trials.

Adrenal Cortex Hormones↗
PubMed1998

Construction of a food-grade multiple-copy integration system for Lactococcus lactis.

A food-grade vector system was developed that allows stable integration of multiple plasmid copies in the chromosome of Lactococcus lactis. The vector consists of the plus origin of replication (Ori+) of the lactococcal plasmid pWV01, the sucrose genes of the lactic acid bacterium Pediococcus pentosaceus PPE1.0 as selectable marker, a multiple-cloning site, and a lactococcal DNA fragment of a well-characterized chromosomal region. The system includes two L. lactis strains, LL108 and LL302, which produce the pWV01 RepA protein essential for replication of the Ori+ vectors. These helper strains allow the construction and isolation of the replicating form of the integration plasmids from a homologous background. Single-crossover integration of the plasmids in L. lactis MG1363 resulted in amplifications to a level of approximately 20 copies/chromosome after selection of the transformants on medium containing sucrose as the only fermentable sugar. The amplifications were stable under selective growth conditions. In glucose-containing medium a limited loss of integrated plasmid copies was detected at a rate of (7.5-15) x 10(-2) copies per generation. One strain, MG124, was isolated that had retained 11 integrated copies after a period of 120 generations of non-selective growth. These results show that the single-cross-over integration system described here represents a simple procedure for the engineering of stable food-grade strains carrying multiple copies of a gene of interest.

Blotting, Southern↗