A fast and reproducible method to estimate left atrial volume using cardiac computed tomography.

PURPOSE: The purpose of this study was to do better than the simple maximal axial area (MAreaax) and to validate simple, fast and robust orthogonal methods for determining the left atrium volume (LAV) with cardiac CT (CCT). MATERIAL AND METHODS: A total of 60 patients who underwent CTT were retrospectively included. There were 30 men and 30 women with a mean age of 59±12 (SD) years (range: 27-80 years), using four methods to assess LAV beyond the MAreaax: two orthogonal methods requiring the measurements of axial, coronal and sagittal planes of 3 diameters LAV3diam or 3 area LAV3Areas; Area-length biplane method LAVbiplane; and volumetric method LAVvolumetric using a semi-automated tool that served as the reference standard. The orthogonal methods were applied on contrast-enhanced (IV+) and unenhanced (IV-) CCT images. Comparisons were performed using Pearson correlation test (r) and Bland-Altman analysis. Inter- and intra-observer variability were assessed using intraclass correlation coefficient (ICC) with a two-way mixed-effects model. RESULTS: On IV+ CCT, LAVbiplane, LAV3diam-IV+, LAV3Areas-IV+ underestimated LAV (-15±1.99mL; -21±1.37mL; -15±1.98mL; all P<0.001). LAV3diam-IV+, LAV3Areas-IV+ better correlated with reference standard (r=0.97 and 0.98) than LAVbiplane (r=0.79) as well as MAreaax (r=0.90). Estimating LAV on IV- further showed high correlation against the reference (r=0.93 and 0.95 for LAV3diam-IV- and LAV3Areass-IV-, respectively). Intra- and inter-observer ICC increased from LAVvolumetric (2.43% and 3.09%); LAV3Areas-IV+ (3.04 and 3.30%); LAV3Areas-IV-(3.34 and 4.23%), LAV3diam-IV+ (3.36 and 5.11%); LAV3diam-IV- (5.16 and 6.90%); to LAVbiplane (9.65 and 10.28%). CONCLUSIONS: Better than MAreaax, orthogonal methods using either diameter or surface are fast and reproducible methods to assess LAV on CCT when performed with or without intravenous administration of contrast material.

The significance of computer-assisted surgery in avoiding double-barrel fibula grafts in reconstruction of the horizontal mandibular ramus.

It is recognized that the mandibular reconstruction with free flap allows for both functional and aesthetic restoration. The flap must be used for reconstruction of a smooth lower mandibular contour and also the alveolar bone to allow dental rehabilitation. The double-barrel fibula free flap does respond well to these imperatives, but it increases risk of vascular complications. To overcome these risks, we propose a single-barrel fibula free flap, positioned at the alveolar level and a customized 3D implant, designed by computer-assisted surgery, at the basilar level. The interest of the single-barrel fibula associated with a customized 3D implant compared to the double-barrel fibula free flap is twofold: it decreases the vascular risks associated with the plication of the second pedicle and allows patients to have a pedicle of greater length. It also allows them to have a good cortical bone placed in the alveolar bone to rehabilitate the mastication function.

Nutritional status of Vietnamese outpatients with chronic obstructive pulmonary disease.

BACKGROUND: Nutritional screening and assessment is not currently part of routine clinical practice in Vietnam. Therefore, the present study aimed to investigate the utility of the commonly used methods for identifying malnutrition in outpatients with chronic obstructive pulmonary disease (COPD). METHODS: A cross-sectional pilot study and a larger retrospective study were carried out in outpatients with COPD who were attending a respiratory clinic in Ho Chi Minh City, Vietnam. Routine clinical data were collected [body mass index (BMI), forced expiratory volume in 1 s (FEV1 )]. Nutritional screening and assessment were performed using the Malnutrition Screening Tool (MST) and Subjective Global Assessment (SGA) as the gold standard to diagnose malnutrition. RESULTS: In total, 393 outpatients had documented BMI and 29 were prospectively assessed using SGA: males, n = 25; females, n = 4; mean (SD) age 69.7 (9.6) years; mean (SD) BMI 21.0 (3.4) kg m-2 ; mean (SD) FEV1 percentage predicted 57.0% (19.7%). Malnutrition risk was identified in 20.7% (n = 6) of patients using the MST (38% sensitivity; 94% specificity). However, 45% (n = 13) were diagnosed as malnourished using the SGA (31% mild/moderate; 14% severe). All malnourished patients not identified by the MST had evidence of muscle wasting. BMI had a strong negative correlation with muscle wasting as assessed using the SGA (r = -0.857, n = 28; P < 0.001) and all malnourished patients had a BMI <21 kg m-2 (range 14.6-20.8 kg m-2 , nourished range 20.0-27.6 kg m-2 ). CONCLUSIONS: Malnutrition is common in Vietnamese outpatients with COPD. A BMI threshold of <21 kg m-2 appears to represent a useful and pragmatic cut-off point for identifying outpatients requiring comprehensive nutritional assessment and support.

[Temporo-mandibular ankylosis]. / L'ankylose temporo-mandibulaire.

Ankylosis of the temporomandibular joint is defined as a permanent constriction of the jaws with less than 30mm mouth opening measured between the incisors, occurring because of bony, fibrous or fibro-osseous fusion. Resulting complications such as speech, chewing, swallowing impediment and deficient oral hygiene may occur. The overall incidence is decreasing but remains significant in some developing countries. The most frequent etiology in developed countries is the post-traumatic ankylosis occurring after condylar fracture. Other causes may be found: infection (decreasing since the advent of antibiotics), inflammation (rheumatoid arthritis and ankylosing spondylitis mainly) and congenital diseases (very rare). Management relies on surgery: resection of the ankylosis block in combination with bilateral coronoidectomy… The block resection may be offset by the interposition temporal fascia flap, a costochondral graft or a TMJ prosthesis according to the loss of height and to the impact on dental occlusion. Postoperative rehabilitation is essential and has to be started early, to be intense and prolonged. Poor rehabilitation is the main cause of ankylosis recurrence.

[Temporomandibular disorders and Ehlers-Danlos syndrome, hypermobility type: A case-control study]. / Dysfonction de l'appareil manducateur et syndrome d'Ehlers-Danlos de type hypermobile : étude cas-témoin.

INTRODUCTION: The Ehlers-Danlos syndrome, hypermobility type (EDS-HT) is a rare genetic disease. Diagnosis is based on a combination of clinical criteria described in the classification of Villefranche. Diagnosis is difficult to make because of the lack of specific clinical signs and the absence of genetic testing. The EDS-TH manifests itself manly by musculoskeletal pain and joint hypermobility. Temporomandibular disorders (TMD) are also reported. Our aim was to objectify the presence and to qualify the type of TMD associated with the EDS-HT in order to propose an additional diagnostic argument. MATERIAL AND METHODS: A prospective, monocenter case-control study, comparing a cohort of patients suffering from EDS-HT to a paired control group of healthy volunteers has been conducted. Clinical examination was standardized, including a general questioning, an oral examination and a temporomandibular joint examination following the TMD/RDC (temporomandibular disorders/research diagnostic criteria). RESULTS: Fourteen EDS-HT patients and 58 control patients were examined. The prevalence of TMDs (n=13; 92.9% vs. n=4; 6.9%; P=10(-11)) was significantly higher in the EDS-HT group. TMDs occurring in the EDS-HT group were complex, combining several mechanisms in contrast to the control group, where only one mechanism was found in all the patients (n=13; 92.9% vs. n=0; 0.0%). DISCUSSION: TMDs are strongly associated with RDS-HT. TMDs could therefore be used in the diagnosis of this disease.

[National survey: Avulsion of included wisdom teeth in mandibular orthognathic surgery]. / Enquête nationale : avulsion des dents de sagesse incluses en chirurgie orthognathique mandibulaire.

INTRODUCTION: There is no recommendation concerning wisdom teeth (WT) extraction in mandibular orthognathic surgery. We carried out an investigation among the members of the French Society of Stomatology and Oro-maxillofacial Surgery (SFSCMFCO), in order to evaluate the practices and habits of maxillofacial surgeons in this field. MATERIALS AND METHODS: We emailed the 424 members of the SFSCMFCO with a questionnaire. RESULTS: We obtained 143 feedbacks that could be exploited. In total, 72.5% of practitioners prefer WT to be extracted before performing a bilateral sagittal spilt osteotomy (BSSO). In this case, a period of 6 months between the two surgeries was considered as desirable by more than 70% of the surgeons. In total, 74.6% of the surgeons thought that the presence of WT could make a BSSO more complicated. However, 73.9% of the surgeons would not postpone the BBSO in a patient ready for surgery but with remaining impacted WT. DISCUSSION: A majority of surgeons think that the presence of impacted WT may complicate a BSSO and increases the risk of bad split. Most of the authors recommend extracting the impacted WT 6 months before BSSO at least. However, these potential complications are easy to overcome and don't compromise the final result. Therefore, we think that impacted WT should not delay a BSSO if the orthodontic preparation makes the patient ready for surgery.

[Perception of asymmetry smile: Attempt to evaluation through Photoshop]. / Perception de l'asymétrie du sourire : tentative d'évaluation grâce à Photoshop.

UNLABELLED: In the labial palliative surgery of facial paralysis, it can persist asymmetry smile. OBJECTIVE: Evaluate the impact of an augmentation or reduction of the commissural course on the perception of a smile anomaly, and determine from which asymmetry threshold, the smile is estimated unsightly. MATERIAL AND METHOD: We took a picture of two people with a smile not forced; including one with a "cuspid smile", and the another one with a "Mona Lisa" smile. The pictures obtained were modified by the Photoshop software, to simulate an asymmetry labial smile. The changes were related to the move of the left labial commissure, the left nasolabial furrow, and the left cheek using under-correction and overcorrection, every 4 mm. Three pictures with under-correction and four pictures with over-correction were obtained. These smiles were shown to three groups of five people, which included doctors in smile specialties, doctors in other specialties, and non-doctors. Participants were then asked to indicate on which of the pictures, the smile seemed abnormal. RESULTS: Between -8 mm under-correction, and +8 mm over-correction, the asymmetry of the commissural course does not hinder the perception of smile. CONCLUSION: In the labial palliative surgery of facial paralysis, in the case of persistent asymmetry, there is a tolerance in the perception of "normality" of smile concerning the amplitude of the commissural course going up to 8 mm of asymmetric with under-correction or over-correction.

The paratransgenic potential of Lactobacillus kunkeei in the honey bee Apis mellifera.

The honey bee (Apis mellifera) is a domestic insect of high value to human societies, as a crop pollinator in agriculture and a model animal in scientific research. The honey bee, however, has experienced massive mortality worldwide due to the phenomenon Colony Collapse Disorder (CCD), resulting in alarming prospects for crop failure in Europe and the USA. The reasons for CCD are complex and much debated, but several honey bee pathogens are believed to be involved. Paratransgenesis is a Trojan horse strategy, where endogenous microorganisms are used to express effector molecules that antagonise pathogen development. For use in honey bees, paratransgenesis must rely on a set of criteria that the candidate paratransgenic microorganism must fulfil in order to obtain a successful outcome: (1) the candidate must be genetically modifiable to express effector molecules; (2) the modified organism should have no adverse effects on honey bee health upon reintroduction; and (3) it must survive together with other non-pathogenic bee-associated microorganisms. Lactic acid bacteria (LAB) are common gut bacteria in vertebrates and invertebrates, and some have naturally beneficial properties in their host. In the present work we aimed to find a potential paratransgenic candidate within this bacterial group for use in honey bees. Among isolated LAB associated with bee gut microbiota, we found the fructophilic Lactobacillus kunkeei to be the most predominant species during foraging seasons. Four genetically different strains of L. kunkeei were selected for further assessment. We demonstrated (1) that L. kunkeei is transformable; (2) that the transformed cells had no obvious adverse effect on honey bee survival; and (3) that transformed cells survived well in the gut environment of bees upon reintroduction. Our study demonstrates that L. kunkeei fulfils the three criteria for paratransgenesis and can be a suitable candidate for further research on this strategy in honey bees.

Glucose control and cardiovascular outcomes: reorienting approach.

Cardiovascular disease accounts for nearly 70% of morbidity and mortality in patients with diabetes mellitus. Strides made in diabetes care have indeed helped prevent or reduce the burden of microvascular complications in both type 1 and type 2 diabetes. However, the same cannot be said about macrovascular disease in diabetes. Several prospective trials so far have failed to provide conclusive evidence of the superiority of glycemic control in reducing macrovascular complications or death rates in people with advanced disease or those with long duration of diabetes. There are trends that suggest that benefits are restricted to those with lesser burden and shorter duration of disease. Furthermore, it is also suggested that benefits might accrue but it would take a longer time to manifest. Clinicians are faced with the challenge to decide how to triage patients for intensified care vs less intense care. This review focuses on evidence and attempts to provide a balanced view of the literature that has radically affected how physicians treat patients with macrovascular disease. It also takes cognizance of the fact that the natural course of the disease may be changing as well, possibly related to better overall awareness and possibly improved access to information about better individual healthcare. The review further takes note of some hard held notions about the pathobiology of the disease that must be interpreted with caution in light of new and emerging data. In light of recent developments ADA and EASD have taken step to provide some guidance to clinicians through a joint position statement. A lot more research would be required to figure out how best to manage macrovascular disease in diabetes mellitus. Glucocentric stance would need to be reconsidered, and attention paid to concurrent multifactorial interventions that seem to be effective in reducing vascular outcomes.

Natural antimicrobial peptides from bacteria: characteristics and potential applications to fight against antibiotic resistance.

Because of the emergence of antibiotic-resistant pathogens worldwide, a number of infectious diseases have become difficult to treat. This threatening situation is worsened by the fact that very limited progress has been made in developing new and potent antibiotics in recent years. However, a group of antimicrobials, the so-called bacteriocins, have been much studied lately because they hold a great potential in controlling antibiotic-resistant pathogens. Bacteriocins are small antimicrobial peptides (AMPs) produced by numerous bacteria. They often act toward species related to the producer with a very high potency (at pico- to nanomolar concentration) and specificity. The common mechanisms of killing by bacteriocins are destruction of target cells by pore formation and/or inhibition of cell wall synthesis. Several studies have revealed that bacteriocins display great potential in the medical sector as bacteriocinogenic probiotics and in the clinic as therapeutic agents. In this review, we discuss the emerging antibiotic resistance and strategies to control its dissemination, before we highlight the potential of AMPs from bacteria as a new genre of antimicrobial agents.

Transposition of IS10R in Lactococcus lactis.

AIMS: To characterize the transposition mechanism of the IS-element IS10R and study how this element is involved in gene disruption in Lactococcus lactis. METHODS AND RESULTS: The gene flciA confers immunity against lactococcin A in lactococci. However, the immunity function was lost when flciA was co-expressed with the regulator gene nisR on a plasmid in L. lactis NZ9000. By PCR and DNA sequencing, it was revealed that flciA in immune-negative transformants was disrupted by the IS-element IS10R. Such gene disruption did not occur when flciA was expressed alone nor when the plasmid-located nisR was mutated, suggesting that nisR is directly involved in the transposition. The sequence 5'-CACTTAACC-3', which was found in flciA and at both ends of the inserted IS10R, was identified as target site by site-directed mutagenesis. CONCLUSIONS: IS10R transposes in L. lactis NZ9000 in a nisR-dependent fashion and employs the sequence 5'-CACTTAACC-3' as integration site. SIGNIFICANCE AND IMPACT OF THE STUDY: To our knowledge, this is the first time IS10R and aspects of its transposition are described in the industrial important bacterium L. lactis. The highly controllable insertion of IS10R into a target site might present a great potential as a gene disruption system.

Use of lactobacilli and their pheromone-based regulatory mechanism in gene expression and drug delivery.

Lactobacilli are common microorganisms in diverse vegetables and meat products and several of these are also indigenous inhabitants in the gastro-intestinal (GI) tract of humans and animals where they are believed to have health promoting effects on the host. One of the highly appreciated probiotic effects is their ability to inhibit the growth of pathogens by producing antimicrobial peptides, so-called bacteriocins. Production of some bacteriocins has been shown to be strictly regulated through a quorum-sensing based mechanism mediated by a secreted peptide-pheromone (also called induction peptide; IP), a membrane-located sensor (histidine protein kinase; HPK) and a cytoplasmic response regulator (RR). The interaction between an IP and its sensor, which is highly specific, leads to activation of the cognate RR which in turn binds to regulated promoters and activates gene expression. The HPKs and RRs are built up by conserved modules, and the signalling between them within a network is efficient and directional, and can easily be activated by exogenously added synthetic IPs. Consequently, components from such regulatory networks have successfully been exploited in construction of a number of inducible gene expression systems. In this review, we discuss some well-characterised quorum sensing networks involved in bacteriocin production in lactobacilli, with special focus on the use of the regulatory components in gene expression and on lactobacilli as potential delivery vehicle for therapeutic and vaccine purposes.

Efficient expansion and gene transduction of mouse neural stem/progenitor cells on recombinant fibronectin.

Neural stem/progenitor cells (NSCs) are commonly grown as floating neurospheres in medium containing basic fibroblast growth factor and epidermal growth factor. Under these conditions, about 1% of the cells retain multipotentiality. We developed a protocol based on culture of NSCs in adherence on recombinant fibronectin (rFN) to transduce up to 90% NSCs at a multiplicity of infection of 2 with no need for viral concentration or production of serum-free retroviral supernatants. NSCs grew faster on rFN than as neurospheres on tissue culture plastic and did not lose their stem cell nature or multipotentiality. Furthermore, retroviral-mediated transgene expression was sustained with time in culture and upon differentiation into neurons and astrocytes. These experimental conditions may be utilized to study the function of various genes in NSCs, and to manipulate NSCs for gene and cell therapy of several neurological diseases.

Evidence for dual functionality of the operon plnABCD in the regulation of bacteriocin production in Lactobacillus plantarum.

The regulatory operon (plnABCD) involved in bacteriocin production in Lactobacillus plantarum C11 encodes four different proteins: a cationic prepeptide (PlnA); a histidine protein kinase (PlnB); and two highly homologous response regulators (PlnC and PlnD; over 75% sequence similarity). The mature product of PlnA, plantaricin A, serves as an extracellular pheromone that induces bacteriocin production. The exact roles of plnBCD in bacteriocin production have not been established experimentally. A reporter system containing the gusA gene fused with the plnA promoter was used to study plnABCD. We demonstrated that the plnABCD operon codes for an autoregulatory unit capable of activating its own promoter. Deletion analyses, performed in a heterologous expression host to define the roles of the individual genes, confirmed that both the inducer gene (plnA) and the kinase gene (plnB) are required for autoactivation. Apparently, the latter gene encodes a protein that serves as a receptor for the pheromone peptide. It was also demonstrated conclusively that the two regulators PlnC and PlnD, which have been shown previously to bind specifically to the DNA regulatory repeats of the plnA promoter, possess differential activities on the plnA promoter, with PlnC being much more active than PlnD. The functions of the response regulators were investigated further in the bacteriocin producer strain C11 in order to reveal their roles in bacteriocin production. Surprisingly, the two response regulators display totally opposite functions: although overexpression of plnC activated transcription and bacteriocin production, the overexpression of plnD repressed both processes, thus strongly suggesting that PlnD plays a role in the downregulation of bacteriocin synthesis. To our knowledge, this is the first evidence for a protein involved directly in negative regulation of bacteriocin production, and also it was shown for the first time that two highly homologous response regulators, with opposite functions, are encoded by genes located on the same operon.

Type II secretion by Aeromonas salmonicida: evidence for two periplasmic pools of proaerolysin.

Aeromonas salmonicida containing the cloned gene for proaerolysin secretes the protein via the type II secretory pathway. Here we show that altering a region near the beginning of aerA led to a dramatic increase in the amount of proaerolysin that was produced and that a large amount of the protein was cell associated. All of the cell-associated protein had crossed the cytoplasmic membrane, because the signal sequence had been removed, and all of it was accessible to processing by trypsin during osmotic shock. Enlargement of the periplasm was observed by electron microscopy in overproducing cells, likely caused by the osmotic effect of the very large concentrations of accumulated proaerolysin. Immunogold electron microscopy localized nearly all of the proaerolysin in the enlarged periplasm; however, only half of the protoxin was released from the cells by osmotic shocking. Cross-linking studies showed that this fraction contained normal dimeric proaerolysin but that proaerolysin in the fraction that was not shockable had not dimerized, although it appeared to be correctly folded. Both periplasmic fractions were secreted by the cells; however, the nonshockable fraction was secreted much more slowly than the shockable fraction. We estimated a rate for maximal secretion of proaerolysin from the bacteria that was much lower than the rates that have been estimated for inner membrane transit, which suggests that transit across the outer membrane is rate limiting and may account for the periplasmic accumulation of the protein. Finally, we show that overproduction of proaerolysin inhibited the release of the protease that is secreted by A. salmonicida.

Spinal cord repair with PHPMA hydrogel containing RGD peptides (NeuroGel).

A biocompatible hydrogel of poly[N-(2-hydroxypropyl)methacrylamide] (PHPMA) which includes the cell-adhesive region of fibronectin Arg-Gly-Asp was synthesized and its structure, rheological and dielectric properties were characterized. The ability of a PHPMA-RGD hydrogel to promote tissue regeneration and support axonal outgrowth in the injured adult and developing rat spinal cord was evaluated. The structure of the PHPMA-RGD hydrogel displayed an interconnected porous structure, with viscoelastic properties similar to those of the neural tissue, and conductivity properties due to a peptide group. The polymer hydrogel provided a structural, three-dimensional continuity across the defect, facilitating the migration and reorganization of local wound-repair cells, as well as tissue development within the lesion. Angiogenesis and axonal growth also occurred within the microstructure of the tissue network, and supraspinal axons migrated into the reconstructed cord segment. In addition, the hydrogel induced a reduction of necrosis and cavitation in the adjacent white and gray matter. These polymer hydrogel matrices therefore display the potential to repair tissue defects in the central nervous system by enhancing the development of a tissue equivalent as well as axonal growth across the reconstructed lesion.

Regulation of bacteriocin production in Lactobacillus plantarum depends on a conserved promoter arrangement with consensus binding sequence.

Bacteriocin production in Lactobacillus plantarum C11 is regulated by a three-component signal transduction system comprising a peptide pheromone (PlnA), a histidine protein kinase (PlnB), and two homologous response regulators (RRs; PlnC and PlnD). Both RRs are DNA-binding proteins that bind to promoter-proximal elements in the pln regulon. The binding site for the two regulators consists of two 9-bp direct repeats, that conform to the consensus sequence 5'-TACGTTAAT-3', and the repeats are separated by an intervening 12-bp AT-rich spacer region. In the present work, the plhA promoter was used as a model to evaluate the significance of the binding sequence and conserved promoter arrangement. Point substitutions in the consensus sequence, particularly those in invariant positions, either abolished or significantly reduced binding of PlnC and PlnD. Both regulators bind as homodimers to DNA fragments containing a complete set of regulatory elements, while removal of either repeat, or alterations in the length of the spacer region, significantly weakened binding of both protein dimers. DNase I footprinting demonstrated that PlnC and PlnD both bind to, and protect, the direct repeats. By fusing the plnA promoter region to the beta-glucuronidase (GUS) gene, it was shown that promoter activity is dependent on an intact set of accurately organized repeats. The in vitro and in vivo results presented here confirm the involvement of the repeats as regulatory elements in the regulation of bacteriocin production.

Expression and properties of an aerolysin--Clostridium septicum alpha toxin hybrid protein.

Aerolysin is a bilobal channel-forming toxin secreted by Aeromonas hydrophila. The alpha toxin produced by Clostridium septicum is homologous to the large lobe of aerolysin. However, it does not contain a region corresponding to the small lobe of the Aeromonas toxin, leading us to ask what the function of the small lobe is. We fused the small lobe of aerolysin to alpha toxin, producing a hybrid protein that should structurally resemble aerolysin. Unlike aerolysin, the hybrid was not secreted when expressed in Aeromonas salmonicida. The purified hybrid was activated by proteolytic processing in the same way as both parent proteins and, after activation, it formed oligomers that corresponded to the aerolysin heptamer. Like aerolysin, the hybrid was far more active than alpha toxin against human erythrocytes and mouse T lymphocytes. Both aerolysin and the hybrid bound to human glycophorin, and both were inhibited by preincubation with this erythrocyte glycoprotein, whereas alpha toxin was unaffected. We conclude that aerolysin contains two receptor binding sites, one for glycosyl-phosphatidylinositol-anchored proteins that is located in the large lobe and is also found in alpha toxin, and a second site, located in the small lobe, that binds a surface carbohydrate determinant.

Secretion and properties of the large and small lobes of the channel-forming toxin aerolysin.

Aerolysin is a dimeric protein secreted by Aeromonas spp. that binds to glycosylphosphatidylinositol-anchored receptors on target cells and becomes insertion competent by oligomerizing. The protein comprises two lobes joined by a short arm. The large lobe is thought to be responsible for channel formation, whereas the small lobe is believed to stabilize the dimer, and it may also contain the receptor binding site. We cloned and expressed the DNA for both lobes of the toxin separately and together in A. salmonicida. The large lobe produced alone was secreted, although more poorly than native protein. The small lobe with the arm produced by itself was not secreted. When the large lobe without the arm was co-produced with the small lobe with the arm, both were secreted, and they co-purified as a stoichiometric complex. Analytical ultracentrifugation showed that they form a heterotetramer corresponding to the native dimer. The purified product was nearly as active as aerolysin, but lost activity and became trypsin sensitive above 25 degreesC. The large lobe with the arm was also purified. It was shown to be monomeric, confirming that the small lobe is responsible for dimer stabilization. The large lobe had very low channel-forming activity, although it was correctly processed by trypsin, and it could form stable oligomers. Surprisingly, the large lobe was found to bind to several glycosylphosphatidylinositol-anchored proteins, indicating that it contains at least part of the receptor-binding domain.

Identification of the DNA-binding sites for two response regulators involved in control of bacteriocin synthesis in Lactobacillus plantarum C11.

In Lactobacillus plantarum C11, bacteriocin production has previously been shown to be an inducible process, in which a secreted peptide, produced by the host itself, is involved. The inducing factor, designated plantaricin A (PlnA), is a bacteriocin-like peptide encoded by a gene (plnA) located on the same operon as the genes for a two-component regulatory system (plnBCD). This system consists of a histidine kinase (PlnB) and two response regulators (PlnC,D), and belongs to a recently defined subfamily of two-component regulatory systems, which are activated by secreted peptide pheromones through a quorum-sensing mechanism. We show here that the two response regulators PlnC and PlnD bind specifically to imperfect direct repeats found within the adjacent promoter of the plnABCD operon, and to similar sequences found within the promoter regions of two nearby operons containing bacteriocin structural genes (plnEFI and plnJKLR). Binding of PlnC and PlnD was increased two to three fold in the presence of acetyl phosphate. The results suggest that bacteriocin synthesis in L. plantarum C11 is regulated by the DNA-binding activity of the two response regulators PlnC and PlnD.