The lambda-toxin of Clostridium perfringens type B NCIB10691 was purified by ammonium sulfate precipitation, followed by size exclusion, anion-exchange, and hydrophobic interaction chromatography. The purified toxin had an apparent molecular mass of 36 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The toxin possessed casein-hydrolyzing activity, which was inhibited specifically by metal chelators, indicating that the toxin is a metalloprotease. The gene encoding the lambda-toxin (lam), which was shown by Southern analysis to be located on a 70-kb plasmid, was cloned into Escherichia coli cells. Nucleotide and N-terminal amino acid sequencing revealed that the lam gene encodes a 553-amino-acid protein, which is processed into a mature form, the molecular mass of which was calculated to be 35,722 Da. The deduced amino acid sequence of the mature enzyme contains an HEXXH motif characteristic of zinc metalloproteases and is homologous to other known enzymes belonging to the thermolysin family. The purified toxin degraded various biologically important substances, such as collagen, fibronectin, fibrinogen, immunoglobulin A, and the complement C3 component. It caused an increase in vascular permeability and hemorrhagic edema on injection into the dorsal skin of mice. These results suggest that the toxin contributes to the pathogenesis of histolytic infection by lambda-toxin-producing C. perfringens.
The plc gene, which encodes phospholipase C (a-toxin) of Ckwtridium petfi-ingens, possesses three poly(A) tracts forming an intrinsically curved DNA region immediately upstream of the promoter. The in vivo transcriptional activity of the plasmid-borne plc gene was stimulated by this curved-DNAcontaining sequence, depending on i t s proper finear and rotationaf orientation. The in vitro transcriptional activity of the pic gene was also stimulated by the upstream sequence. In addition, the stimulatory effect of the sequence and the degree of DNA bending were greater at lower temperature, as was demonstrated by both in vitro and in vivo transcription assays, and a gel-mobility assay, respectively. A similar temperature effect was also observed with the chromosomal plc gene. These observations suggest that the upstream DNA curvature per se stimulates the initiation of transcription of the plc gene, possibly through direct contact with RNA polymerase.
Some individuals are at risk of anti-D alloimmunization if they inherit D antigens that are qualitatively and/or quantitatively different than wild-type D. We hypothesized that patients who showed serologically inconsistent, weak, or historically discordant D typing results by microplate direct agglutination (MDA) on NEO or Echo (Immucor, Norcross, GA) might be at risk of carrying RHD allelic variants. The present study was designed to evaluate patients with RHD allelic variants if they presented with weakly reactive D typing results on the NEO or Echo. Patients were selected for RHD genotyping if their specimens showed weak reactivity with either series 4 or series 5 anti-D typing reagent, if the strength of reactivity was ≤1+ on the NEO or Echo, or if historical or current D typing results were discordant with current results. Patients selected for RHD genotyping were also tested by saline tube testing using the same anti-D series 4 and 5 reagents. Genotyping was performed by the Immucor genotyping laboratory in Warren, NJ. Of 80 patients whose samples met study inclusion, 52 (65.0%) were found to have RHD allelic variants. Sixteen patients (20.0%) expressed possible Ceppellini effect reactivity. Most importantly, 51.25 percent of the patients who presented with weakly reactive D typing results by MDA testing on the NEO (≤1+) or Echo (≤1+) had RHD allelic variants that were associated with the potential for anti-D alloimmunization. Laboratories that use MDA testing on the Neo or Echo for D typing should consider that female patients of childbearing age might be at risk of anti-D alloimmunization if they are classified as D+ based on weakly reactive D typing results.
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