Previous studies indicate that action potential duration (APD) alternans is initiated in the endocardial (END) and midmyocardial (MID) regions rather than the epicardium (EPI) in the canine left ventricle (LV). This study examines regional differences in the rate dependence of Ca 2ϩ transient characteristics under conditions that give rise to APD and associated T wave alternans. The role of the sarcoplasmic reticulum (SR) was further evaluated by studying Ca 2ϩ transient characteristics in myocytes isolated from neonates, where an organized SR is poorly developed. All studies were performed in cells and tissues isolated from the canine LV. Isolated canine ENDO, MID, and EPI LV myocytes were either field stimulated or voltage clamped, and Ca 2ϩ transients were measured by confocal microscopy. In LV wedge preparations, increasing the basic cycle length (BCL) from 800 to 250 ms caused alternans to appear mainly in the ENDO and MID region; alternans were not observed in EPI under these conditions. Ca 2ϩ transient alternans developed in response to rapid pacing, appearing in EPI cells at shorter BCL compared with MID and ENDO cells (BCLϭ428 Ϯ 17 vs. 517 Ϯ 29 and 514 Ϯ 21, respectively, P Ͻ 0.05). Further increases in pacing rate resulted in the appearance of subcellular alternans of Ca 2ϩ transient amplitude, which also appeared in EPI at shorter BCL than in ENDO and MID cells. Ca 2ϩ transient alternans was not observed in neonate myocytes. We conclude that 1) there are distinct regional differences in the vulnerability to rate-dependent Ca 2ϩ alternans in dog LV that may be related to regional differences in SR function and Ca 2ϩ cycling; 2) the development of subcellular Ca 2ϩ alternans suggests the presence of intracellular heterogeneities in Ca 2ϩ cycling; and 3) the failure of neonatal cells to develop Ca 2ϩ alternans provides further support that SR Ca 2ϩ cycling is a major component in the development of these phenomena.alternans; calcium ion transients; heterogeneity REPOLARIZATION OR T WAVE ALTERNANS (TWA) is an electrocardiographic (ECG) phenomenon characterized by a beat-to-beat alternation of the amplitude, morphology, and/or polarity of the T wave. Electrical remodeling can occur under a variety of pathophysiological conditions such as acquired and congenital long QT syndromes (33), heart failure (21), and cardiac ischemia (23), producing the conditions for the initiation of arrhythmias (arrhythmogenic substrate). Despite the link between the appearance of alternans and the development of arrhythmias, the mechanism underlying the development of alternans remains unresolved. Previous studies indicate there are large differences in the magnitude of L-type Ca 2ϩ current (I Ca ) and the rapid form of the delayed-rectifier K ϩ current during the long and the short action potentials (APs), suggesting an ionic component to the development of alternans (12,15,18 Refs. 6,16,24,and 38). A prominent transient outward K ϩ current (I to )-mediated phase 1 often present in epicardial (EPI) and midmyocardial (MID) cel...
Bacteria communicate with each other to regulate cell density-dependent gene expression via a quorumsensing (QS) cascade. In Pseudomonas aeruginosa, two known QS systems, las and rhl, control the expression of many factors that relate to virulence, pathogenicity, and biofilm development. Microarray studies of the las and rhl regulons led to our hypothesis that a complicated hierarchy in the QS regulon is composed of multiple transcriptional regulators. Here, we examined a QS-regulated gene, vqsR, which encodes a probable transcriptional regulator with a putative 20-bp operator sequence (las box) upstream. The transcriptional start site for vqsR was determined. The vqsR promoter was identified by examining a series of vqsR promoter-lacZ fusions. In addition, an Escherichia coli system where either LasR or RhlR protein was expressed from a plasmid indicated that the las system was the dominant regulator for vqsR. Electrophoretic mobility shift assays (EMSA) demonstrate that purified LasR protein binds directly to the vqsR promoter in the presence of 3O-C 12 -HSL. Point mutational analysis of the vqsR las box suggests that positions 3 and 18 in the las box are important for vqsR transcription, as assayed with a series of vqsRp-lacZ fusions. EMSA also shows that positions 3 and 18 are important for binding between the vqsR promoter and LasR. Our results demonstrate that the las system directly regulates vqsR, and certain nucleotides in the las box are crucial for LasR binding and activation of the vqsR promoter.Pseudomonas aeruginosa is an opportunistic human pathogen and a major cause of nosocomial infections. It infects immunocompromised individuals and patients with the pulmonary disorder cystic fibrosis (10). The expression of many P. aeruginosa virulence factors is controlled by a regulatory mechanism known as quorum sensing (QS) (5, 23). QS is a form of intercellular communication that allows bacteria to coordinate gene expression in response to cell density. In many gramnegative bacteria, QS is accomplished by the production of diffusible signal molecules in the form of acyl homoserine lactones, or autoinducer, and a transcriptional regulatory protein (R protein) that serves as a signal receptor and transcriptional regulator. Both autoinducer and R protein are produced at basal levels when the population density is low. As cell density increases, autoinducer molecules accumulate. As a threshold concentration of autoinducer is reached, it forms a complex with the R protein. The R protein-autoinducer complex is able to regulate its target genes (9).In P. aeruginosa, two primary QS systems, las and rhl, have been identified. The las system includes LasI and LasR, and the rhl system includes RhlI and RhlR. LasI catalyzes the synthesis of the N-(3-oxododecanoyl)-L-homoserine lactone (3O-C 12 -HSL) signal molecule, which binds to the transcriptional regulator protein LasR. RhlI catalyzes the synthesis of N-butanoyl-L-homoserine lactone (C 4 -HSL), which binds to the RhlR protein. These two systems form a hierarchic...
To identify antigens specific for the filamentous form of Candida albicans, a combinatorial phage display library expressing human immunoglobulin heavy and light chain variable regions was used to select phage clones capable of binding to the surfaces of viable C. albicans filaments. Eight distinct phage clones that bound specifically to filament surface antigens not expressed on blastoconidia were identified. Single-chain antibody variable fragments (scFv) derived from two of these phage clones (scFv5 and scFv12) were characterized in detail. Filament-specific antigen expression was detected by an indirect immunofluorescence assay. ScFv5 reacted with C. dubliniensis filaments, while scFv12 did not. Neither scFv reacted with C. glabrata, C. parapsilosis, C. rugosa, C. tropicalis, or Saccharomyces cerevisiae grown under conditions that stimulated filament formation in C. albicans and C. dubliniensis. Epitope detection by the two scFv was sensitive to proteinase K treatment but not to periodate treatment, indicating that the cognate epitopes were composed of protein. The antigens reactive with scFv5 and scFv12 were extractable from the cell surface with Zymolyase, but not with sodium dodecyl sulfate (SDS) and 2-mercaptoethanol, and migrated as polydisperse, high-molecular-weight bands on SDS-polyacrylamide gel electrophoresis gels. The epitopes were detected on clinical specimens obtained from infants with thrush and urinary candidiasis without passage of the organisms on laboratory media, confirming epitope expression in human infection. The availability of a monoclonal immunologic reagent that recognizes filaments from both C. albicans and C. dubliniensis and another specific only to C. albicans adds to the repertoire of potential diagnostic reagents for differentiation between these closely related species.
The generation of new antibodies for diagnostic applications using phage display could greatly decrease the time and expense of new assay development but, to be effective, the process must yield antibodies with desired specificity and affinity comparable to those obtained by monoclonal antibody technology. In order to evaluate the ability of the phage selection process to yield antibodies with the desired specificity and affinity, a family of anti-phenobarbital antibodies were cloned as scFvs and Fabs and displayed on M13 gene III fusion proteins. All of the antibodies are derived from similar germline VL and VH genes and exhibit extensive sequence homology except in VH CDR3. Despite these similarities, the range of panning efficiencies was observed to vary by two orders of magnitude for expressed scFvs. Unexpectedly, the scFv with the highest panning efficiency has the lowest affinity. In competitive panning experiments this scFv is preferentially isolated over higher affinity antibodies. This scFv expresses high levels of soluble binding protein while higher affinity scFvs express lower levels of protein or nonfunctional protein. These results suggest that the efficiency of functional expression of scFv proteins can greatly influence the type of antibody selected by phage display. The range of panning efficiency for functional Fabs was significantly less (four-fold) than that observed for scFvs and did not correlate to the expression level of the secreted proteins. Based on the results of the Fabs examined, it is concluded that the expression properties of Fabs may not exhibit the extent of variability observed for scFvs when displayed and use of an Fab architecture may provide an advantage over scFv architecture in the selection process. The feasibility of selecting against undesirable cross-reactivities has also been demonstrated by simple modification of phage panning conditions. These combined results support the concept of obtaining antibodies with desirable specificity and affinity for diagnostic applications through the use of phage display.
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