No abstract

Two monoclonal antibodies (116831 and H5TS) differed in their indirect immunofluorescence reactivity when tested against 14 strains of Lyme disease spirochetes. Strains were bound by both antibodies, by H6831 or H5TS alone, or by neither. Western blot and immunoprecipitation studies revealed that the determinants of both antibodies were associated with abundant proteins with apparent subunit molecular weights of ca. 34,000 (34K-range proteins). The following results indicated that the 34K-range proteins were exposed on the surface of the spirochetes. (i) Antibody H6831 agglutinated the spirochetes; (ii) immune electron microscopy showed that the H6831 determinant was associated with the outer membrane; (iii) radiolabeled H6831 bound to live organisms; and (iv) proteases effectively removed the 34K-range proteins from intact cells. With their demonstrated variability and exposure on the surface, the 34K-range proteins may contribute to the serotype specificity of Lyme disease spirochetes. A spirochete which was first discovered in Ixodes dammini ticks (12) is the etiological agent of Lyme disease (LD) (8, 9, 21) and probably of the related disorders erythema chronicum migrans (1, 13; K. Weber, G. Schierz, B. Wilske, and V. Preac-Mursic, Yale J. Biol. Med., in press) and tickassociated meningoradiculitis (Bannwarth's syndrome) (1, 17, 18) as well. The LD complex of syndromes has been reported from the continents of Europe, North America, and Australia (2, 22, 23). These LD spirochetes have to date been isolated from ticks, mammals, and humans in the United States (3, 8-10, 12, 21) and from ticks collected in areas of Europe where erythema chronicum migrans and tick-associated meningoradiculitis are endemic (5; R. Ackerman and B. Skoldenberg, personal communications). Although these spirochete isolates have shown considerable homogeneity in their polyacrylamide gel electrophoresis (PAGE) protein profiles (5, 7) and in the reactivity of a common 31,000-molecular-weight protein with a monoclonal antibody (H5332 [7]), there also were indications of signifi

Ixodid tick-associated spirochetes have been implicated as the etiological agents of Lyme disease. We raised a murine monoclonal antibody (H5332) against a spirochete, strain B31, isolated from Ixodes dammini ticks. In indirect immunofluorescence assays and western blot analyses, H5332 reacted with whole cells or isolated components of not only strain B31 but also spirochetes isolated from Ixodes ricinus ticks, a field mouse, a raccoon, and patients with Lyme disease. In contrast, H5332 did not bind to representative borreliae, treponemes, and leptospires. Using indirect immunofluorescence assays and immune electron microscopy, we found the H5332 determinant to be diffusely distributed over the surface of prefixed spirochetes but to be aggregated in patches when the organisms were incubated with H5332 and a second ligand before fixation. Radioimmunoprecipitation and western blot studies revealed the H5332 determinant to be either on or tightly associated with an abundant outer membrane protein with an apparent subunit molecular weight of 31,000.

For researchers doing qualitative research, interviews are a commonly used method. Data collected through interviews can be recorded through field notes, transcripts, or tape recordings. In the literature, there is a debate regarding which of these recording methods should be used. There are issues of reliability, cost (time and money), loss of data, among others. Technology plays a pivotal role in this debate. Indeed, new technologies (e.g., direct coding) are often seen as potential replacements for older technologies (e.g., transcripts), which leads to a debate that is based on an evolution narrative (from field notes, to transcripts, to working from tape recordings). This article argues that a combination narrative should be considered where combination is better than substitution. Moreover, combining the advantages of field notes, transcripts, and working from tape recordings without accumulating each method's disadvantages is possible because of new technology. To support this argument, two technological tools (OneNote and SmartPen) are presented as a way to increase the effectiveness, efficiency, and economy of qualitative data management.

Ca(2+)/calmodulin-dependent protein kinases II (CaMKII) have important functions in regulating cardiac excitability and contractility. In the present study, we examined whether CaMKII regulated the transient outward K(+) current (I(to)) in whole-cell patch-clamped human atrial myocytes. We found that a specific CaMKII inhibitor, KN-93 (20 micromol/L), but not its inactive analog, KN-92, accelerated the inactivation of I(to) (tau(fast): 66.9+/-4.4 versus 43.0+/-4.4 ms, n=35; P<0.0001) and inhibited its maintained component (at +60 mV, 4.9+/-0.4 versus 2.8+/-0.4 pA/pF, n = 35; P<0. 0001), leading to an increase in the extent of its inactivation. Similar effects were observed by dialyzing cells with a peptide corresponding to CaMKII residues 281 to 309 or with autocamtide-2-related inhibitory peptide and by external application of the calmodulin inhibitor calmidazolium, which also suppressed the effects of KN-93. Furthermore, the phosphatase inhibitor okadaic acid (500 nmol/L) slowed I(to) inactivation, increased I(sus), and inhibited the effects of KN-93. Changes in [Ca(2+)](i) by dialyzing cells with approximately 30 nmol/L Ca(2+) or by using the fast Ca(2+) buffer BAPTA had opposite effects on I(to). In BAPTA-loaded myocytes, I(to) was less sensitive to KN-93. In myocytes from patients in chronic atrial fibrillation, characterized by a prominent I(sus), KN-93 still increased the extent of inactivation of I(to). Western blot analysis of atrial samples showed that delta-CaMKII expression was enhanced during chronic atrial fibrillation. In conclusion, CaMKII control the extent of inactivation of I(to) in human atrial myocytes, a process that could contribute to I(to) alterations observed during chronic atrial fibrillation.

In immunofluorescence assays monoclonal antibody H9724 recognized eight species of the spirochetal genus Borrelia but not representatives of the genera Treponema, Leptospira, and Spirochaeta. We examined the reactivity of H9724 against subcellular components of Borrelia hermsii, an agent of relapsing fever, and B. burgdorferi, the cause of Lyme disease. H9724 bound to isolated periplasmic flagella of the two borreliae. In Western blots the antibody reacted with the predominant protein in flageliar preparations from B. hermsii and B. burgdorfferi; the apparent molecular weights of these flagellins were 39,000 and 41,000, respectively.

No abstract

No abstract