Comparison of LightCycler PCR, rapid antigen immunoassay, and culture for detection of group A streptococci from throat swabs.

We compared the performance characteristics of a real-time PCR method, the LightCycler Strep-A assay (Roche Applied Science, Indianapolis, Ind.), to those of a rapid antigen immunoassay, the Directigen 1-2-3 Group A Strep Test kit (BD Diagnostic Systems, Sparks, Md.), and a standard culture method for detection of group A streptococci (GAS) from 384 throat swabs. The LightCycler PCR produced more positive results (n = 58) than either culture (n = 55) or the Directigen immunoassay (n = 31). The results of the LightCycler PCR and the Directigen method were independently compared to the results of the accepted "gold standard," bacterial culture. The sensitivities, specificities, and positive and negative predictive values for this comparison were as follows: for the Directigen method, 55, 99, 97, and 93%, respectively; for the LightCycler PCR, 93, 98, 88, and 99%, respectively. In no case was a throat swab positive by both the LightCycler PCR and the Directigen method but negative by culture. The medical histories of patients whose throat swabs were negative by culture but positive by either the LightCycler PCR (n = 7) or the Directigen method (n = 1) were reviewed. All of these patients had signs or symptoms compatible with GAS disease, and therefore, all of these discordant positive results (along with positive results by either the Directigen method or the LightCycler PCR that agreed with the culture results) were counted as true positives for statistical analysis. For this analysis, the LightCycler PCR detected more true-positive results than the culture method (58 versus 55 swabs); however, this difference was not statistically significant (P = 0.5465). In contrast, statistically significantly more true-positive results occurred by culture than by the Directigen method (55 versus 31 swabs; P < 0.0001) and by the LightCycler PCR than by the Directigen method (58 versus 31 swabs; P < 0.0001). The LightCycler PCR is a suitable stand-alone method for the detection of GAS from throat swabs. Additionally, this method requires less than half the personnel time and the procedure can be completed in considerably less time ( approximately 1 h) than our standard approach (up to 2 days) for detection of GAS in throat swabs (i.e., testing by the Directigen method with negative results verified by culture).

A riparian wildlife habitat evaluation scheme developed using GIS.

To evaluate riparian habitat for wildlife, we used a geographic information system (GIS) that prioritized individual streams (for acquisition or management) by habitat ranking. We demonstrate this methodology for the Vermilion River basin in east-central Illinois, USA. Three data sets were used to evaluate land cover encompassing 300 m on either side of the streams: (1) the US Geological Survey's land use and land cover information (LUDA), (2) land cover manually digitized from the National High Altitude Photography (NHAP) program, and (3) Landsat Thematic Mapper (TM) data classified into land cover. Each of 30 tributaries in the study area was ranked for habitat according to the data contained in each data set, and results were compared. Habitat ranking schemes were devised and analysis performed for three species guilds: forest, grassland, and mixed successional species. TM and NHAP each differentiated habitat scores (for forest, grassland, and mixed successional guilds) among tributaries in a similar and suitable way, while LUDA was not suitable, due to the coarse resolution of the data. Overall, it was shown that the methodology is suitable to rank streams based on riparian habitat quality. Even though more work is needed to test and verify the method, the project has shown the potential for such techniques to assist in evaluating, tracking, and improving the management of riparian wildlife resources. The method can easily be applied over large areas such as states if TM-based land cover and stream data are available.

Apoptotic proteins Reaper and Grim induce stable inactivation in voltage-gated K+ channels.

Drosophila genes reaper, grim, and head-involution-defective (hid) induce apoptosis in several cellular contexts. N-terminal sequences of these proteins are highly conserved and are similar to N-terminal inactivation domains of voltage-gated potassium (K+) channels. Synthetic Reaper and Grim N terminus peptides induced fast inactivation of Shaker-type K+ channels when applied to the cytoplasmic side of the channel that was qualitatively similar to the inactivation produced by other K+ channel inactivation particles. Mutations that reduce the apoptotic activity of Reaper also reduced the synthetic peptide's ability to induce channel inactivation, indicating that K+ channel inactivation correlated with apoptotic activity. Coexpression of Reaper RNA or direct injection of full length Reaper protein caused near irreversible block of the K+ channels. These results suggest that Reaper and Grim may participate in initiating apoptosis by stably blocking K+ channels.

Tissue-specific alternative splicing of Shaker potassium channel transcripts results from distinct modes of regulating 3' splice choice.

Alternative splicing of precursor RNA enables a single gene to encode multiple protein isoforms with different functional characteristics and tissue distributions. Differential splicing of Drosophila Shaker (Sh) gene transcripts regulates the tissue-specific expression of kinetically distinct potassium ion channels throughout development. Regulation of Sh alternative splicing is being examined in germline transformants using lacZ as a reporter gene. P-element constructs were generated in which one or both of the two mutually exclusive Sh 3' acceptor sites were positioned in the same translational reading frame as the lacZ coding sequences. The constructs were introduced into the germline and the transgenic animals examined for tissue-specific beta-galactosidase expression patterns. Some tissues exhibit "promiscuous" splicing; these tissues are competent to splice to either 3' acceptor even when both are present on the same pre-mRNA. In other tissues splice choice results from competition between the two 3' sites; these tissues can splice to either site when it is the only available 3' acceptor, but when given a choice will splice to only one of the two 3' acceptors. In some tissues, splicing occurs exclusively at only one of the 3' acceptor sites; these tissues are not competent to splice to one of the sites even if it is the only 3' acceptor present on the pre-mRNA. These results suggests that multiple, distinct regulatory modes are operating to control tissue-specific alternative splicing of Sh 3' domains and are discussed in terms of potential underlying mechanisms for regulating the tissue-specific expression of alternatively spliced genes.

Tissue-specific alternative splicing of hybrid Shaker/lacZ genes correlates with kinetic differences in Shaker K+ currents in vivo.

Alternative splicing of the Shaker (Sh) locus of Drosophila generates several transcripts with divergent 5' and 3' domains that produce kinetically distinct K+ currents in Xenopus oocytes. Although suggestive that alternative splicing may be involved in generating K+ channel diversity, clear tissue-specific differences in the distribution of particular Sh gene products have not been demonstrated. Using lacZ as a reporter gene for accurate splicing of variable Sh3' domains, we observe differences in beta-galactosidase expression patterns in transgenic animals that indicate both temporal and spatial regulation of 3' splice choice. The differences in 3'splice choice can account for variation in recovery kinetics of Sh-encoded K+ currents recorded in adult flies. The results indicate that tissue-specific expression of functionally distinct Sh K+ channels is regulated, in part, at the level of pre-mRNA splicing and implicate sequences in or around the 3' splice sites in regulating the choice of 3' domain.

Functional expression of Shaker K+ channels in cultured Drosophila "giant" neurons derived from Sh cDNA transformants: distinct properties, distribution, and turnover.

Expression of transgenic Shaker (Sh) channels has not previously been examined in Drosophila neurons. We studied K+ current by whole-cell recording in cultured "giant" neurons derived from germline transformants. Independent lines were generated by using a P-element vector, in which transcription of the 29-4 cDNA, one of the Sh splicing variants (Iverson and Rudy, 1990), was under the control of a heat shock (HS)-inducible promoter. Transformants in wild-type and two different Sh mutant backgrounds all exhibited an HS-inducible, A-type K+ current that was characterized by a much slower recovery from inactivation and a higher sensitivity to 4-aminopyridine than native K+ currents of Sh 29-4 currents expressed in Xenopus oocytes. Despite similarities in the kinetic and pharmacological properties of the HS-induced current in all backgrounds examined, host-dependent differences in the peak current amplitude have been consistently observed between multiple lines of 29-4 ShM and 29-4 Sh120 that might reflect differential channel subunit assembly in different hosts. Isolation of the novel 29-4 currents allowed determination of the channel turnover rate in cultured neurons. These currents persisted for up to 3 d or more, comparable with the durations previously reported for Na+ and Ca2+ channels. Surprisingly, the percentage of cells expressing inactivating K+ currents remained approximately the same with or without HS induction, suggesting that some mechanisms exist to restrict functional expression of inactivating K+ channels, including transgenic Sh channels and those not encoded by the Sh locus, to certain types of neurons.

Evaluation of two rapid antigen assays, BioStar Strep A OIA and Pacific Biotech CARDS O.S., and culture for detection of group A streptococci in throat swabs.

Two rapid methods, BioStar Strep A OIA (OIA; BioStar, Inc., Boulder, Colo.), an optical immunoassay, and CARDS O.S. (O.S.; Pacific Biotech, Inc., San Diego, Calif.), a color immunochromographic assay, and two culture methods, one with 5% sheep blood agar (SBA) and one with Todd-Hewitt broth (TH; Remel, Lenexa, Kans.), were evaluated for use in the detection of Streptococcus pyogenes from pharnygeal swabs. Seven hundred forty-six double swabs (Culturette II) were processed, with OIA and SBA culture performed on one swab and O.S. and SBA culture performed on the other swab. The pledget from the Culturette II was incubated overnight in TH and was subcultured onto SBA for an additional 48 h in ambient air. All beta-hemolytic streptococci from culture were tested by a direct fluorescent-antibody test (Difco Laboratories, Detroit, Mich.). Specimens with discordant fluorescent-antibody test and rapid test results were also tested by using the Streptex latex agglutination reagent (Murex Diagnostics Limited, Dartford, England). The results obtained by all testing methods were compared with a combined test result ("gold standard"), which was defined as any positive culture detected by the SBA or TH culture methods and confirmed by Streptex latex agglutination or, in the case of negative results by both culture methods, a concomitant positive result by OIA and O.S. antigen testing. Sensitivity and specificity results for each of the methods were as follows, respectively: OIA, 81.0 and 97.5%; O.S., 74.4 and 99.0%; SBA culture, 92.3 and 98.3%; and TH culture, 86.4 and 100%. Both OIA and O.S. are suitable screening methods for detecting S. pyogenes directly from throat swabs but are of insufficient sensitivity to eliminate the need for backup cultures for specimens with negative OIA and O.S. results.

Simplified correction of outflow obstruction after mitral valve replacement.

Left ventricular outflow tract obstruction after mitral valve replacement may occur when a retained native anterior leaflet prolapses between prosthetic struts. Existing reports of left ventricular outflow tract obstruction by this mechanism lack emphasis on its surgical treatment. We obtained definitive relief of left ventricular outflow tract obstruction by transaortic exposure, division, and partial excision of the obstructing leaflet. This approach minimizes the complexity and potential morbidity of the correction.

Tandem linkage of Shaker K+ channel subunits does not ensure the stoichiometry of expressed channels.

Shaker K+ channels are multimeric, probably tetrameric proteins. Substitution of a conserved leucine residue to valine (V2) at position 370 in the Drosophila Shaker 29-4 sequence results in large alterations in the voltage dependence of gating in the expressed channels. In order to determine the effects of this mutation in hybrid channels with a fixed stoichiometry of V2 and wild-type (WT) subunits we generated cDNA constructs of two linked-monomeric subunits similar to the tandem constructs previously reported by Isacoff, E. Y., Y. N. Jan, and L. Y. Jan. (1990. Nature (Lond.). 345:530-534). In addition, we constructed a tandem cDNA containing a wild-type subunit and a truncated nonfunctional subunit (Sh102) that suppresses channel expression. We report that the voltage-dependence of the channels produced with WT and V2 subunits varied significantly with the order of the subunits in the construct (WT-V2 or V2-WT), while the WT-Sh102 construct yielded currents that were much larger than expected. These results suggest that the tandem linkage of Shaker subunits does not guarantee the stoichiometry of the expressed channel proteins.

Platelet activating factor as a proinflammatory mediator in acetic-induced colitis in the rat.

Platelet activating factor (PAF) induces neutrophilia and produces a variety of gastrointestinal lesions. The role of PAF as a proinflammatory mediator was examined by measuring the production of PAF and the efficacy of the PAF antagonists WEB 2086 and Ro 24-0238 in acetic acid (HOAc)-induced colitis. PAF levels within the colonic mucosa, as measured by radioimmunoassay, increased from 259 +/- 119 ng/mg in control tissue, to 616 +/- 266 ng/mg in HOAc inflamed tissue. The accumulation of neutrophils within the mucosa was decreased by 53 +/- 10% by pretreatment with 3 mg/kg WEB 2086, and by 43 +/- 11% by 3 mg/kg Ro 24-0238. PAF-induced neutrophilia had no effect on the severity of HOAc-induced colitis, therefore, PAF my be involved in sustaining the chronic inflammation of colitis.

A role for hydrophobic residues in the voltage-dependent gating of Shaker K+ channels.

A leucine heptad repeat is well conserved in voltage-dependent ion channels. Herein we examine the role of the repeat region in Shaker K+ channels through substitution of the leucines in the repeat and through coexpression of normal and truncated products. In contrast to leucine-zipper DNA-binding proteins, we find that the subunit assembly of Shaker does not depend on the leucine heptad repeat. Instead, we report that substitutions of the leucines in the repeat produce large effects on the observed voltage dependence of conductance voltage and prepulse inactivation curves. Our results suggest that the leucines mediate interactions that play an important role in the transduction of charge movement into channel opening and closing.

Antimicrobial proteins in human unstimulated whole saliva in relation to each other, and to measures of health status, dental plaque accumulation and composition.

Saliva antimicrobial proteins may interact in a common system to influence the oral ecology. Clinical studies of antimicrobial protein action thus may require a multiple-protein approach. Multivariate statistical methods have been used to describe possible patterns of interaction for lysozyme, lactoferrin, salivary peroxidase and secretory IgA in stimulated parotid saliva. However, oral microbes are most likely to encounter antimicrobial proteins in mixed resting saliva. Relationships among levels of lysozyme, lactoferrin, salivary peroxidase, and secretory IgA therefore were investigated in whole saliva from 216 subjects, and an attempt made to relate interperson variation in those proteins to differences in health and status, and dental plaque accumulation and composition. All proteins were significantly (alpha = 0.05) correlated with each other (r = 0.38-0.52, p less than 0.001). There was only one axis of common variation among proteins, and that axis was significantly correlated (p less than 0.001) with total protein (r = 0.84) and flow rate (r = -0.56). That pattern deviated from the previous finding that proteins of acinar origin tended to vary independently from proteins of ductal origin in stimulated parotid saliva. The difference between parotid and whole saliva may reflect constitutive secretion of all proteins at low levels of stimulation. Common variation of unstimulated saliva proteins suggests that antimicrobial actions can be compared in subjects at population extremes. There were no significant associations between antimicrobial proteins in whole saliva and measures of health status or plaque accumulation. However, the proportions of Streptococcus sanguis were significantly correlated with lysozyme (r = -0.26), lactoferrin (r = -0.34), peroxidase (r = -0.30), total protein (r = -0.37), flow rate (r = 0.24) and principal-components scores (r = -0.33) in a subset of subjects (n = 85) where commercial biochemical tests were used to supplement species identification by colony morphology. Those findings may indicate that saliva antimicrobial proteins can affect the composition of dental plaque.

Shaker K+ channel subunits from heteromultimeric channels with novel functional properties.

A large number of related genes (the Sh gene family) encode potassium channel subunits which form voltage-dependent K+ channels by aggregating into homomulitimers. One of these genes, the Shaker gene in Drosophila, generates several products by alternative splicing. These products encode proteins with a constant central region flanked by variable amino and carboxyl domains. Coinjection of two Shaker RNAs with different amino or different carboxyl ends into Xenopus oocytes produces K+ currents that display functional properties distinct from those observed when each RNA is injected separately, indicating the formation of heteromultimeric channels. The analysis of Shaker heteromultimers suggests certain rules regarding the roles of variable amino and carboxyl domains in determining kinetic properties of heteromultimeric channels. Heteromultimers with different amino ends produce currents in which the amino end that produces more inactivation dominates the kinetics. In contrast, heteromultimers with different carboxyl ends recover from inactivation at a rate closer to that observed in homomultimers of the subunit which results in faster recovery. While this and other recent reports demonstrate that closely related Sh family proteins form functional heteromultimers, we show here that two less closely related Sh proteins do not seem to form functional heteromultimeric channels. The data suggest that sites for subunit recognition may be found in sequences within a core region, starting about 130 residues before the first membrane spanning domain of Shaker and ending after the last membrane spanning domain, which are not conserved between Sh Class I and Class III genes.

The role of the divergent amino and carboxyl domains on the inactivation properties of potassium channels derived from the Shaker gene of Drosophila.

Several products generated from the Drosophila Shaker gene by alternative splicing predict a group of similar proteins with an identical central and variable amino and carboxyl domains. We have constructed 9 Sh cDNAs combining 3 different 5' domains with 3 different 3' domains. RNA transcribed from 6 of these cDNAs induce K+ currents in Xenopus oocytes. All currents share similar properties of voltage dependence, potassium selectivity, and block by 4-AP, TEA, and charybdotoxin. These properties presumably result from a channel core formed by the identical central region of the proteins. The currents differ in macroscopic inactivation kinetics. Five RNAs induced K+ currents which inactivate, each at distinct rates, during short depolarizations. The sixth RNA induces a current that essentially does not inactivate unless depolarized for many seconds. This raises the possibility that Sh may encode nontransient as well as transient K+ currents. Analysis of currents produced by the various combinations suggests that the divergent amino domains influence the stability of a first, nonabsorbing, inactivated state. This results in striking differences in the probability of channel reopening, as observed in single-channel recordings, of those channels with identical carboxyl but different amino domains. Furthermore, based on macroscopic analysis of the currents, we suggest that the primary role of the carboxyl domains is to influence the relative stability between the first and a second inactivated state. The second inactivated state is essentially absorbing, and recovery from this state is very slow. The observed differences in the rates of recovery from inactivation of channels containing different carboxyl domains reflect differences in the rates at which they enter this second inactivated state.

Porcine heparin increases postoperative bleeding in cardiopulmonary bypass patients.

One hundred thirteen patients undergoing cardiopulmonary bypass were randomly assigned to receive either bovine or porcine heparin. Heparin was infused at 4.5 mg/kg during bypass and administered at the lesser of 70 units/kg or 5000 units/dose at 12-hour intervals postoperatively. Platelet counts decreased to 45% of preoperative levels during the first 3 days postoperatively (porcine, 44 +/- 13%, n = 50; bovine, 46 +/- 15%), but returned to preoperative levels by the seventh postoperative day. The average blood loss in the porcine heparin group significantly exceeded that of the bovine heparin group (porcine, 1350.7 +/- 727.8 ml; bovine, 1059.6 +/- 381.0 ml; p less than .01). Consequently, the platelet transfusion requirement was greater in the porcine heparin group (porcine, 1.7 +/- 3.9 units; bovine, 0.5 +/- 1.7 units; p less than .05); however, blood and blood component (with the exception of platelets) administration was not significantly different between the two groups. The four patients taking anticoagulants or antiinflammatory agents in the porcine group required a mean of 8.5 units of red blood cells (RBC) plus supplemental platelets. The seven such patients in the bovine group received a mean of 3.0 units of RBC and no platelets. Thus, the use of porcine heparin resulted in a generalized increase in postoperative bleeding with increased management problems in patients undergoing cardiopulmonary bypass.

Omental pedicle grafting in the treatment of postcardiotomy sternotomy infection.

Postcardiotomy sternal infection occurred in 20 (2%) of 1007 patients undergoing cardiac surgery between September 1985 and December 1987, a 10-fold increase over the preceding 33 months (4 [0.24%] of 1627 patients). Cultures were sterile in 5 patients and yielded staphylococci in 12 and a variety of bowel organisms in 3. The cause for the increased occurrence of sternal wound infection is unclear after multivariate analysis, although infections have precipitously dropped subsequent to changing to cefuroxime sodium antibiotic prophylaxis. Treatment has evolved to appropriate antibiotics and early débridement of involved sternum and cartilage. Rewiring the sternum is not attempted. If gross purulence is not present, primary closure is accomplished using muscle flaps (2 patients) or omental pedicle grafts (17 patients). In the presence of gross purulence, the wound is packed open for 5 days and then closed in the above fashion. Two patients required skin grafts for primary closure. The omental pedicle flap is preferred due to simplicity and improved coverage of the sternal defect inferiorly. Nineteen patients healed primarily. A superficial wound infection was drained in 1 patient. Midline incisional hernias developed in 3 muscular patients. Omentum is now harvested through a left subcostal incision. Hospital stay was under 2 weeks in 13 patients. One death occurred due to multisystem failure prior to completion of wound closure. In our experience, early sternal débridement and omental pedicle grafting with primary closure is appropriate therapy for postcardiotomy sternotomy infections. The presence of gross purulence may require 5 days of open packing prior to omental grafting. No significant complications occurred, and mortality was low. A left subcostal incision for omental harvesting is utilized to avoid the occurrence of delayed incisional hernias.