The PCR products were digested with BamHI and XhoI and inserted between the same restriction sites of the plasmid pET-26b+ to form pETSN, pETSB and pETSC, respectively. The three recombined plasmids were transformed into E. coli. BL21 (DE3) plys competent cells and plated onto Luria–Bertani (LB) plates with 30 μg mL−1 kanamycin. DNA sequences were sequenced by the Nanjing GenScript Biotechnology Co., Ltd. The plasmids pETSN, pETSB and pETSC which contain the correct gene sequence of the wild-type enzyme, served as the templates for DNA family shuffling. Initially, the target gene of the enzymes was amplified using CT99021 mw PCR with

the primers described above. The PCR products were purified and subjected to DNase I digestion to generate random fragments according to the method described by Suen et al. (2004). The procedures for DNA shuffling were performed based on the method described by Stemmer (1994) with minor modifications. The digested products were subjected to 2% agarose gel electrophoresis, and DNA fragments of 50–100 bp were recovered for primer-less DNA assembly. Pfu DNA polymerase was used in the PCR method to reduce new mutations that may be introduced into the parental selleck chemicals gene sequences. The gradient PCR program of the primer-less PCR was 94 °C for 5 min, followed by 45 cycles of 94 °C for 30 s, 55 °C for 45 s, 50 °C for 45 s, 47 °C for 45 s, 44 °C

for 45 s, and 72 °C for 2 min. The products of primer-less PCR were purified and diluted 10 times for PCR using the primers PNB and PNX (Table 1). After heating for 5 min at 94 °C, the reaction program was 94 °C for 1 min, 56.6 °C for 1 min, 72 °C for 2 min (30 cycles), with a final extension of 72 °C for 10 min. The mutated PCR products were purified, digested with BamHI and XhoI, and inserted into the pET-26b+ vector, which was cut using the same enzymes, followed by the transformation into E. coli BL21(DE3)pLysS competent cells to obtain the mutant library. The mutant library was primarily screened on LB plates containing 30 μg mL−1 of kanamycin and 2%

(w/v) skim milk (Tange et al., 1994). After 24–48 h of cultivation at 37 °C, colonies that formed larger clear however zones were isolated using sterile toothpicks and transferred to a 5-mL liquid LB culture containing 30 μg mL−1 kanamycin. The bacterial isolates were cultured at 37 °C for 12 h, induced for 4 h by the addition of isopropyl-β-d-thiogalactopyranoside (IPTG) and centrifuged. The pellets of bacteria were resuspended and diluted to OD600 nm = 0.1 with 100 mM phosphate buffer (pH 8.0). The cells were lysed by sonication, and the crude enzyme fibrinolytic activity in the supernatant was assayed using the fibrin plate method (Astrup & Mullertz, 1952). Those colonies showing higher fibrinolytic activity compared to wild-type NK were selected as the parents for the next round of shuffling.

DART Virology Group: P. Kaleebu (Co-Chair), D. Pillay (Co-Chair), V. Robertson, D. Yirrell, S. Tugume, M. Chirara, P. Katundu, N. Ndembi, F. Lyagoba, D. Dunn, R. Goodall and A. McCormick. DART Health

Economics Group: A. Medina Lara (Chair), S. Foster, J. Amurwon, B. Nyanzi Wakholi, J. Kigozi, L. Muchabaiwa and M. Muzambi. Trial Steering Committee: I. Weller (Chair), A. Babiker (Trial Statistician), S. Bahendeka, M. Bassett, SCH727965 datasheet A. Chogo Wapakhabulo, J. Darbyshire, B. Gazzard, C. Gilks, H. Grosskurth, J. Hakim, A. Latif, C. Mapuchere, O. Mugurungi, P. Mugyenyi; Observers: C. Burke, S. Jones, C. Newland, S. Rahim, J. Rooney, M. Smith, W. Snowden and J.-M. Steens. Data and Safety Monitoring Committee: A. Breckenridge (Chair), A. McLaren selleck chemicals llc (Chair-deceased), C. Hill, J. Matenga, A. Pozniak and

D. Serwadda. Endpoint Review Committee: T. Peto (Chair), A. Palfreeman, M. Borok and E. Katabira. Sources of support: the DART trial is funded by the UK Medical Research Council, the UK Department for International Development (DFID), and the Rockefeller Foundation. First-line drugs for NORA were provided by GlaxoSmithKline and Boehringer Ingelheim. Additional support for viral load and resistance assays in NORA was provided by GlaxoSmithKline. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript Author contributions C.F.G., A.G.B., P.M., J.H.D., D.M.G., P.M., C.K., F.S., A.R. designed the NORA study which P.M., C.K. and F.S. ran. A.S.W. conducted analyses and wrote the first draft of the paper with C.F.G., D.M.G., J.H.D. and A.G.B. All authors contributed Cepharanthine to interpretation of the data, revised the manuscript critically, and approved the final version. No author has a conflict of interest. “
“The aim of the study was to gain more insight into the relationship between transmitted singletons found at HIV diagnosis by population sequencing and the possible presence of clinically relevant viral minorities containing additional resistance mutations. We studied the viral quasispecies and therapy response in 10 individuals with transmitted

single nucleoside reverse transcriptase inhibitor (NRTI)-related resistance mutations as detected by population sequencing. Ultra-deep pyrosequencing did not reveal additional drug-resistance mutations in nine of 10 patients. In these nine patients, no breakthrough with resistant viruses was observed despite the use of low genetic nonnucleoside reverse transcriptase inhibitor (NNRTI)-based regimens in the majority of patients. These data suggest that viral minority variants containing additional resistance mutations may be rare in patients with transmitted NRTI singletons in the Netherlands. Larger studies are required to confirm these findings and to determine the therapeutic consequences. “
“1st Ed , (xvi) + 286 pp , paperback, USD67.95 , ISBN 978-0-7295-3884-8 , Sydney, Churchill Livingstone, Australia : Daniel Ellis and Matthew Hooper , 2010 .

In contrast, toxicity can

occur when an interaction leads to increased antiretroviral concentrations or the patient receives a higher dose than the correct one. Resistance or toxicity is more likely to occur when the error is extended in time or when the error has not been resolved before the patient’s discharge. Some authors have confirmed that HAART-related errors are common in hospitalized patients and that admission of an HIV-infected patient by a physician not specialized in infectious diseases could be a risk factor for drug-related problems [4]. The aims of this study were to identify and describe HAART-related www.selleckchem.com/products/FK-506-(Tacrolimus).html errors in medication prescribed to HIV-infected patients admitted to a tertiary teaching hospital and Ivacaftor to determine the degree of acceptance of the pharmacist’s interventions. We conducted an observational, prospective, 1-year study (between 1 January and 31 December 2007). Twice a week (on Tuesday and Thursday),

a pharmacy resident trained in HIV pharmacotherapy and supported by a staff infectious diseases pharmacist identified patients aged at least 18 years who had been admitted to the Hospital Clinic (a 750-bed tertiary teaching hospital in Barcelona, Spain) and prescribed HAART. A list was made of all inpatients who were prescribed antiretroviral drugs. Admissions made on Fridays, at weekends and on Mondays were recorded on Tuesday afternoon. Admissions made on Tuesdays, Wednesdays and Thursdays were recorded on Thursday afternoon. The following data were recorded for all patients: age, gender, risk factors Thiamine-diphosphate kinase for HIV infection, admitting service, serum creatinine level and liver function (serum albumin, total bilirubin, transaminases, and international normalized ratio). For those patients with an altered creatinine value (>1.2 mg/dL), the glomerular filtration rate was calculated using the Cockcroft–Gault

equation [5]. For those patients with any abnormal liver function test result, the admission report was checked to determine whether they had cirrhosis, in which case the Child–Pugh score [6,7] was also recorded. Concomitant medication was reviewed twice weekly to check for drug–drug interactions. HAART errors were classified as follows: contraindicated or not recommended drug–drug combinations, incorrect or incomplete antiretroviral regimen, omitted dose, incorrect dose (not matching the outpatient prescription), lack of dose reduction for renal or hepatic impairment and incorrect schedule [8]. In Spain, HIV-infected patients pick up their antiretroviral medication in the outpatient pharmacy unit of the hospital that they attend for care. Therefore, it was easy for us to determine the patient’s HAART regimen.

Colonies were scored after a 48-h incubation at 28 °C. In antioxidant protection tests, a reactive oxygen species (ROS) scavenger viz. 1.0 M glycerol selleck kinase inhibitor or 10 mM pyruvate (Patikarnmonthon et al., 2010) was added to bacterial cultures 10 min before heat treatment. All experiments were repeated independently three times. The exponential cultures of X. campestris pv. campestris wild-type and katA-katG double-mutant strains (Jittawuttipoka et al., 2009) were subjected to heat shock at 37 °C for 15 min. Cells were collected by centrifugation at 5000 g for 10 min for total RNA preparation. RT-PCR was carried out to synthesize cDNA as described

previously (Jittawuttipoka et al., 2010). Reverse transcription reaction was performed using 5 μg total RNA, the

RevertAid™ M-MuLV Reverse transcriptase Kit (Fermentas), and random hexamers according to the manufacturer’s recommendation. The specific primer pairs for heat shock genes were BT3194 (5′CCACCAAGGGTGAAGTCG3′)-BT3195 (5′CGCAGCACCTTGTACTCG3′) for groES, BT3190 (5′ATGGCGAGAAGCAGTTCG3′)-BT3191 (5′CGAGGTCGACAGCTCGAT3′) for dnaK, and BT3188 (5′AGCACTACGGCGAAGACG3′)-BT3189 (5′GTCGCGGTGGTACAGGTC3′) this website for hptG. The primer pair for the 16S rRNA gene, which was used as the normalizing gene, was BT2781 (5′GCCCGCACAAGCGGTGGAG3′)-BT2782 (5′ACGTCATCCCCACCTTCCT3′). Real-time PCR was conducted using 20 ng cDNA, a specific primer pair, and SYBR® green PCR Master Mix (Applied Biosystems), and run on an Applied Biosystems StepOne Plus under the following conditions: denaturation at 95 °C for 30 s, annealing at 60 °C for 30 s, and extension at 72 °C for 30 s, for 40 cycles. To monitor the level of the katA transcript MG-132 concentration in the ahpC mutant and the wild-type strains, the ahpC-specific primers BT2684 (5′CGCAGCGTCTCGGTGACG3′)-BT2685 (5′AGTGGAAGACGCCGCTGA3′) were used in the real-time RT-PCR reactions under the following conditions: 40 cycles of denaturation

at 95 °C for 30 s, annealing at 55 °C for 20 s, and extension at 72 °C for 30 s. Relative expression analysis was carried out using stepone software v2.1 and expressed as folds of expression relative to the level of an X. campestris pv. campestris wild type grown under untreated conditions. Experiments were repeated independently three times. Flow cytometric analysis was performed as described previously (Fuangthong et al., 2011). Exponential-phase cultures of X. campestris pv. campestris were washed twice with a phosphate-buffer saline (PBS) solution and resuspended in PBS to yield a cell density of 104 cell mL−1. The cell suspension (500 μL) was mixed with 1 μL of 2 mg mL−1 dihydrorhodamine-123 (DHR) (Molecular Probe) before heat treatment for at 45 °C for 2 min.

Colonies were scored after a 48-h incubation at 28 °C. In antioxidant protection tests, a reactive oxygen species (ROS) scavenger viz. 1.0 M glycerol BTK inhibitor nmr or 10 mM pyruvate (Patikarnmonthon et al., 2010) was added to bacterial cultures 10 min before heat treatment. All experiments were repeated independently three times. The exponential cultures of X. campestris pv. campestris wild-type and katA-katG double-mutant strains (Jittawuttipoka et al., 2009) were subjected to heat shock at 37 °C for 15 min. Cells were collected by centrifugation at 5000 g for 10 min for total RNA preparation. RT-PCR was carried out to synthesize cDNA as described

previously (Jittawuttipoka et al., 2010). Reverse transcription reaction was performed using 5 μg total RNA, the

RevertAid™ M-MuLV Reverse transcriptase Kit (Fermentas), and random hexamers according to the manufacturer’s recommendation. The specific primer pairs for heat shock genes were BT3194 (5′CCACCAAGGGTGAAGTCG3′)-BT3195 (5′CGCAGCACCTTGTACTCG3′) for groES, BT3190 (5′ATGGCGAGAAGCAGTTCG3′)-BT3191 (5′CGAGGTCGACAGCTCGAT3′) for dnaK, and BT3188 (5′AGCACTACGGCGAAGACG3′)-BT3189 (5′GTCGCGGTGGTACAGGTC3′) SAHA HDAC for hptG. The primer pair for the 16S rRNA gene, which was used as the normalizing gene, was BT2781 (5′GCCCGCACAAGCGGTGGAG3′)-BT2782 (5′ACGTCATCCCCACCTTCCT3′). Real-time PCR was conducted using 20 ng cDNA, a specific primer pair, and SYBR® green PCR Master Mix (Applied Biosystems), and run on an Applied Biosystems StepOne Plus under the following conditions: denaturation at 95 °C for 30 s, annealing at 60 °C for 30 s, and extension at 72 °C for 30 s, for 40 cycles. To monitor the level of the katA transcript Thymidylate synthase in the ahpC mutant and the wild-type strains, the ahpC-specific primers BT2684 (5′CGCAGCGTCTCGGTGACG3′)-BT2685 (5′AGTGGAAGACGCCGCTGA3′) were used in the real-time RT-PCR reactions under the following conditions: 40 cycles of denaturation

at 95 °C for 30 s, annealing at 55 °C for 20 s, and extension at 72 °C for 30 s. Relative expression analysis was carried out using stepone software v2.1 and expressed as folds of expression relative to the level of an X. campestris pv. campestris wild type grown under untreated conditions. Experiments were repeated independently three times. Flow cytometric analysis was performed as described previously (Fuangthong et al., 2011). Exponential-phase cultures of X. campestris pv. campestris were washed twice with a phosphate-buffer saline (PBS) solution and resuspended in PBS to yield a cell density of 104 cell mL−1. The cell suspension (500 μL) was mixed with 1 μL of 2 mg mL−1 dihydrorhodamine-123 (DHR) (Molecular Probe) before heat treatment for at 45 °C for 2 min.

The decrease in SICI observed with large peaks in the PTSH and large MEPs suggests that when the conditioning stimulus is weak (0.6–0.7 RMT), the depression of the corticospinal selleck monoclonal humanized antibody inhibitor volley has little effect on motoneuron discharge: the weakly depressed corticospinal inputs after SICI are still sufficient to make the motoneurons discharge. Therefore, the combination of the two methods

(PSTH and MEP studies) suggests that (1) low-threshold cortical neurons activated at low TMS intensities are not sensitive to SICI, (2) the distribution of inhibitory inputs in cortical networks is non-linear and (3) the conditioning stimulus has to be > 0.7 RMT to depress the corticospinal volleys sufficiently and to avoid BVD-523 chemical structure the saturation at motoneuron level that would prevent the evaluation of SICI using the difference between conditioned and test responses. Although the non-invasive techniques used in humans can only provide indirect electrophysiological data, it has been possible (1) to give further evidence for linear input–output properties of cortico-motoneuronal networks (Devanne et al., 1997) and (2) to give the first evidence for non-linear summation of inhibitory inputs in neural networks controlling pyramidal cell discharge in the

human primary motor cortex. To our knowledge, this is the first time that the input–output properties of cortical networks have been studied under physiological conditions (both in humans and in animals). These results are important for the understanding of synaptic integration at cortical level and summation at motoneuron

level in studies using TMS: synaptic integration at cortical and spinal levels should be taken into account in interpreting the effects of TMS. In addition, this study provides further insights into the neural mechanisms underlying plasticity in awake humans. Intrinsic plasticity in layer V cortical neurons has been demonstrated in animal preparations in vivo (Paz et al., 2009), but a change in the relative recruitment gain of inhibitory interneurons has been proposed to participate in long-term potentiation of pyramidal cells only using a computational model (Marder & ADP ribosylation factor Buonomano, 2004). Given the non-linear summation of inhibitory inputs at cortical level, a change in the recruitment gain of inhibitory interneurons can strongly influence pyramidal cell excitability. Such a mechanism should be taken into account in studies using techniques recently developed to investigate TMS-induced plasticity in humans (e.g. repetitive TMS and paired-associative stimulation). We thank Prof. David Burke (Sydney University) for reading and commenting upon the manuscript. The study was supported by UPMC Université Paris 6, Assistance Publique-Hôpitaux de Paris (AP-HP), Institut pour la Recherche sur la Moelle Epinière (IRME), and INSERM. L.S.G. was supported by a grant from UPMC Université Paris 6 (Ministère de l’Enseignement Supérieur et de la Recherche).

Placenta and umbilical cord blood were obtained at delivery and infant blood was obtained within 48 h of delivery. mtDNA content was determined for each specimen. Nuclear [subunit IV of cytochrome c-oxidase http://www.selleckchem.com/products/epz-6438.html (COX IV)]- and mitochondrial (COX II)-encoded polypeptides of the oxidative phosphorylation enzyme cytochrome c-oxidase were quantified in cord and infant blood. Placental mitochondria malondialdehyde (MDA) concentrations were measured as a marker of oxidative

stress. Twenty HIV-positive/HIV-exposed and 26 control mother–infant pairs were enrolled in the study. All HIV-infected women and their infants received ART. Placental MDA concentration and mtDNA content in placenta and cord blood were similar between groups. The

cord blood COX II:IV ratio was lower in the HIV-positive group than in the controls, whereas the infant peripheral blood mtDNA content was higher in the HIV-exposed infants, but the infant peripheral blood COX II:IV ratio was similar. selleckchem No infant had clinical evidence of mitochondrial disease or acquired HIV infection. In multivariable regression analyses, the significant findings in cord and infant blood were both most associated with HIV/ART exposure. HIV-exposed infants showed reduced umbilical cord blood mitochondrial enzyme expression with increased infant peripheral blood mitochondrial DNA levels, the latter possibly reflecting a compensatory mechanism to overcome HIV/ART-associated mitochondrial toxicity. Strategies implemented for HIV-infected pregnant women and HIV-exposed infants, especially combination antiretroviral therapy (ART) given to women during pregnancy, have dramatically decreased the risk of mother-to-child transmission (MTCT) [1]. The vast majority of infants

do not exhibit any clinically apparent toxicity associated with this in utero ART exposure, and therefore Phenylethanolamine N-methyltransferase the benefit of reduced MTCT far outweighs the possible detrimental effects in the infant. However, there is still uncertainty about deleterious mitochondrial effects in ART-exposed infants, based on a number of previous animal and human studies [2–10]. The first report in 1999 from Blanche et al. detailed eight cases of perinatally nucleoside reverse transcriptase inhibitor (NRTI)-exposed, noninfected children with hyperlactataemia who exhibited neurological and developmental sequelae consistent with mitochondrial dysfunction [4]. The same group of investigators also described 12 perinatally NRTI-exposed children in a cohort of 2644 with motor abnormalities, seizures, and cognitive developmental delays, which were often associated with abnormal magnetic resonance imaging (MRI) results and/or significant hyperlactataemia [5]. The 18-month incidence for mitochondrial dysfunction was 0.26% in these ART-exposed children, compared with 0.01% for paediatric neuro-mitochondrial diseases in the general population.

Placenta and umbilical cord blood were obtained at delivery and infant blood was obtained within 48 h of delivery. mtDNA content was determined for each specimen. Nuclear [subunit IV of cytochrome c-oxidase EPZ-6438 cell line (COX IV)]- and mitochondrial (COX II)-encoded polypeptides of the oxidative phosphorylation enzyme cytochrome c-oxidase were quantified in cord and infant blood. Placental mitochondria malondialdehyde (MDA) concentrations were measured as a marker of oxidative

stress. Twenty HIV-positive/HIV-exposed and 26 control mother–infant pairs were enrolled in the study. All HIV-infected women and their infants received ART. Placental MDA concentration and mtDNA content in placenta and cord blood were similar between groups. The

cord blood COX II:IV ratio was lower in the HIV-positive group than in the controls, whereas the infant peripheral blood mtDNA content was higher in the HIV-exposed infants, but the infant peripheral blood COX II:IV ratio was similar. buy Rapamycin No infant had clinical evidence of mitochondrial disease or acquired HIV infection. In multivariable regression analyses, the significant findings in cord and infant blood were both most associated with HIV/ART exposure. HIV-exposed infants showed reduced umbilical cord blood mitochondrial enzyme expression with increased infant peripheral blood mitochondrial DNA levels, the latter possibly reflecting a compensatory mechanism to overcome HIV/ART-associated mitochondrial toxicity. Strategies implemented for HIV-infected pregnant women and HIV-exposed infants, especially combination antiretroviral therapy (ART) given to women during pregnancy, have dramatically decreased the risk of mother-to-child transmission (MTCT) [1]. The vast majority of infants

do not exhibit any clinically apparent toxicity associated with this in utero ART exposure, and therefore Fluorometholone Acetate the benefit of reduced MTCT far outweighs the possible detrimental effects in the infant. However, there is still uncertainty about deleterious mitochondrial effects in ART-exposed infants, based on a number of previous animal and human studies [2–10]. The first report in 1999 from Blanche et al. detailed eight cases of perinatally nucleoside reverse transcriptase inhibitor (NRTI)-exposed, noninfected children with hyperlactataemia who exhibited neurological and developmental sequelae consistent with mitochondrial dysfunction [4]. The same group of investigators also described 12 perinatally NRTI-exposed children in a cohort of 2644 with motor abnormalities, seizures, and cognitive developmental delays, which were often associated with abnormal magnetic resonance imaging (MRI) results and/or significant hyperlactataemia [5]. The 18-month incidence for mitochondrial dysfunction was 0.26% in these ART-exposed children, compared with 0.01% for paediatric neuro-mitochondrial diseases in the general population.

Primarily because of the lack of large-scale clinical evidence, the NICE recommendations were formulated in the absence of any consideration of the possible benefits of certain classes of antihypertensive agents in improving BI 6727 cognition. In the light of the NICE statement above about the absolute difference between ACEIs/AIIAs and CCBs being small, the conclusions of the current review may warrant reconsideration of the

guidelines with reference to: the use of ACEI in the elderly; the recommended preference for brain-penetrability of ACEIs; and the preference of AIIAs over ACEIs. A reconsideration of the use of ACEIs or AIIAs in black patients may also be warranted, albeit not as monotherapy for hypertension. Whether there are ethnic differences in any cognitive responses to ACEIs or AIIAs has yet to be explored, but there is a strong possibility that the cardiovascular and psychological effects are brought about by different mechanisms; hence such ethnic differences may not be the case. Note that the same is true for the use of ACEIs

in heart failure where the NICE guidelines make no reference to differential use in different ethnic groups. There has recently been a call for more clinical trials in the area of hypertension control and dementia in www.selleckchem.com/products/gdc-0068.html the very elderly,[64] and there may also be a need to investigate ethnic differences in any observed drug effects. To return to the title of this review, and its relevance to prescribing practice and patient counselling, it is still unclear which comes first: non-adherence to antihypertensive medication or impaired cognition. There is, however, evidence that antihypertensive medicines, in particular brain-penetrating ACEIs and AIIAs, may reduce the cognitive decline associated with hypertension, and may even improve cognition independent of any cardiovascular effect. Non-adherence to the medication might therefore be predicted to have an adverse effect on cognition.

On the other hand, good adherence to the antihypertensive medication is likely to improve control of blood pressure but also improve cognition, having the ‘positive feedback’ effect of further maintaining the good adherence to medication. Regarding patient Monoiodotyrosine counselling, therefore, not only should patients be told of the benefits of adherence to antihypertensive therapy in terms of the decreased risk of stroke, myocardial infarct and heart failure, but they should also be informed of the possible beneficial effects in terms of decreased prevalence of dementia and Alzheimer’s disease. The Author declares that he has no conflicts of interest to disclose. This review received no specific grant from any funding agency in the public, commercial or not-for-profit sectors. “
“Objectives The aim was to investigate patients’ perceptions and understanding on the appropriate use of non-prescription ibuprofen.

(Thirup et al., 2000), and thus change the nutrient turnover patterns. Conversely, bacteria with secondary metabolite production will resist predation better, which is a serious problem with artificially introduced bacteria (Ekelund & Rønn, 1994). Our results demonstrate that metabolite-producing Pseudomonas affect some protozoan groups more than others and that the most mobile protozoan groups are the most vulnerable. Hence, when considering administration of bacteria to protect plants against

fungi, it is preferable to use bacteria with membrane-bound metabolites as protozoa can better cope with them, and, in nature, the protozoa can avoid them simply by moving to another location. The Danish Research

Council for Technology and Innovation grant no. 23-04-0089 financed FXR agonist the project. Mette Vestergaard and Trine Koch, Biological selleck Institute, Copenhagen University kindly provided us with H. vermiformis and B. designis UJ, respectively. C. Keel provided P. fluorescens CHA0. “
“The use of antisense oligodeoxyribonucleotides (asODNs) to inhibit gene function has proven to be an extremely powerful tool for establishing gene–function relationships. Diffusion limitations imposed by the thick peptidoglycan layer of Gram-positive bacteria have proven difficult to overcome for permeability of asODNs. Typically, introduction of the asODN is achieved by cloning the antisense sequence into a vector downstream of an inducible promoter and transforming this clonidine construct into the cell of interest. In this study, we report that

the use of the streptococcolytic enzyme zoocin A facilitated entry of phosphorothioate oligodeoxyribonucleotides (PS-ODNs) into Streptococcus mutans, such that the degree of phenotypic response (cell growth inhibition) observed was sequence specific and correlated with the amount of zoocin A (R2=0.9919) or PS-ODN (R2=0.9928) used. Quantitative reverse transcriptase PCR was used to demonstrate that only the expression of the target gene against which the PS-ODN was designed was affected. We believe that the use of an appropriate bacteriolytic enzyme to facilitate entry of asODNs into bacterial cells provides a method that will be generally useful in the study of gene regulation in Gram-positive bacteria. Use of antisense oligodeoxyribonucleotides (asODNs) as a means of controlling gene expression in bacteria is proving to be an extremely powerful tool for establishing gene–function relationships and has proven particularly valuable where the gene being examined is essential for cell function (Baev et al., 1999; Harth et al., 2002; Wang & Kuramitsu, 2003). In many bacteria, antisense RNA is a natural gene-expression regulatory process that enables highly specific regulation of selected gene products (Brantl, 2002). asODNs usually consist of 10–30 target-specific nucleotides that are complementary to their target mRNA.