Rapid and effective detection of anthrax spores in soil by PCR

AIMS: To detect Bacillus anthracis DNA from soil using rapid and simple procedures. METHODS AND RESULTS: Various amounts of B. anthracis Pasteur II spores were added artificially to 1 g of soil, which was then washed with ethanol and sterile water. Enrichment of the samples in trypticase soy broth was performed twice. A DNA template was prepared from the second enrichment culture using a FastPrep instrument. The template was then used for nested and real-time polymerase chain reaction (PCR) with B. anthracis-specific primers, to confirm the presence of B. anthracis chromosomal DNA and the pXO1/pXO2 plasmids. CONCLUSIONS: One cell of B. anthracis in 1 g of soil could be detected by nested and real-time PCR. The usefulness of the PCR method using field samples was also confirmed. SIGNIFICANCE AND IMPACT OF THE STUDY: The results indicate that this could be a useful method for detecting anthrax-spore contaminated soil with high sensitivity. Its application could have great impact on the progress of epidemiological surveillance.     

Suppressors of temperature-sensitive mutations in a ribosomal protein gene, rpsL (S12), of Escherichia coli K12

Summary Temperature-sensitive (ts) mutations were isolated within a ribosomal protein gene (rpsL) of Escherichia coli K12. Mutations were mapped by complementation using various transducing phages and plasmids carrying the rpsL gene, having either a normal or a defective promoter for the rpsL operon. One of these mutations, ts118, resulted in a mutant S12 protein which behaved differently from the wild-type S12 on CM-cellulose column chromatography. Suppressors of these ts mutations were isolated and characterized; one was found to be a mutation of a nonribosomal protein gene which was closely linked to the RNAase III gene on the E. coli chromosome. This suppressor, which was recessive to its wild-type allele, was cloned into a transducing phage and mapped finely. A series of cold-sensitive mutations, affecting the assembly of ribosomes at 20°C, was isolated within the purL to nadB region of the E. coli chromosome and one group, named rbaA, mapped at the same locus as the suppressor mutation, showing close linkage to the RNAase III gene.     

Appearance of spore coat protein in the cell extracts of Bacillus subtilis asporogenic mutants.

By use of the antigen-antibody techniques we have studied whether asporogenic mutants of Bacillus subtilis can synthesize the spore coat protein. Antibody specific to spore coat protein was prepared and used to demonstrate that the spore coat protein was synthesized at the early stage of sporulation. We report here that asporogenic mutants synthesize the spore coat protein.     

Antibodies introduced into living cells by red cell ghosts are functionally stable in the cytoplasm of the cells.

The function and fate of antibodies introduced into living cells by red cell ghosts were studied using CRM 176 (a mutant diphtheria toxin having lower toxicity than the wild-type) and antibody against fragment A of diphtheria toxin. IgG labeled with iodine and FITC was found in the cytoplasm of the recipient cells. When about 1500 molecules of anti-fragment A antibody (rabbit IgG) were introduced into diphtheria toxin-sensitive Vero cells or FL cells, these cells became resistant to the toxin and formed normal colonies. It was calculated from the survival of cells without anti-fragment A IgG under these conditions that about 300 molecules of fragment A-176 were transferred to the cells. These results showed that the antigen-antibody reaction took place in living cells as effectively as in a cell-free system. The functional stability of antibody IgG in cells was examined by exposing Vero cells containing a subminimal amount of anti-fragment A IgG (about 1000 molecules) to the toxin for 2 hr at various times after the introduction of anti-fragment A IgG. More than 50% of the initial activity of the antibody to neutralize toxin still remained even after incubation of the cells at 37°C for 20 hr. The same degree of stability was also demonstrated using iodine-labeled specific anti-fragment A IgG. The IgG recovered from the recipient cells after various times of incubation at 37°C retained its full ability to bind to fragment A-conjugated Sepharose 4B, although the total amount of IgG associated with the cells decreased about 50% in 24 hr.     

Degradation of rat cardiac myofibrils and myofibrillar proteins by a myosin-cleaving protease.

The degradation of rat cardiac myofibrils and their constituent proteins with a myosin-cleaving protease was studied. Electrophoretograms of the digestion products of myofibrils showed that myosin,M-protein, C-protein, and troponin were degraded, but actin and tropomyosin were not. Degradation of these constituents resulted in losses of the Mg2+-ATPase activity and its Ca2+-sensitivity of myofibrils. Incubation of myofibrils with the protease induced the release of alpha-actinin without degradation. Susceptibilities of myosin, actin, troponin, and alpha-actinin purified from rat and pig hearts to the protease were essentially identical to those of the assembled forms in myofibrils. Although the purified tropomyosin was readily degraded into five fragments with the protease, the tropomyosin assembled in myofibrils and actin-tropomyosin complex were insusceptible to the protease. Digestion of myosin in the filamentous state with the protease resulted in the disappearance of myosin heavy chain and light chain 2, producing two fragments having molecular weights of 130,000 and 94,000 which originated from the degradation of heavy chain. The Ca2+- and EDTA-ATPase activities of the degradation products remained unchanged during incubation for 22 h. The actin-activated ATPase activity of myosin was reduced by 30% during incubation for 6 h, and recovered to the original level on adding actin to give a ratio of actin to myosin of 2:1. The pH optima for degradation of myosin in the soluble and filamentous states were 8.5 and 7.0, respectively. The results indicate that cardiac myosin in the filamentous state was more readily degraded with the protease than the myosin in the soluble state.     

Reaction of fungal amine oxidase with beta-bromoethylamine.

beta-Br-ethylamine is both a substrate and an irreversible inhibitor of amine oxidase from Aspergillus niger. The enzyme catalyzes the nonoxidative elimination of HBr from beta-Br-ethylamine to form acetaldehyde. beta-Br-ethylamine meets several criteria for an irreversible substrate analog or suicide inhibitor. 1) It inactivates the oxidized enzyme, but not the reduced enzyme. 2) The Michaelis constant for beta-Br-ethylamine in the elimination reaction showed a similar magnitude to that of the related constant found when the haloamine acted as an inhibitor. 3) The enzyme was protected from the inactivation by the co-existence of the substrate. 4) Inactivation with beta-Br-[14C]ethylamine resulted in the incorporation of radioactivity corresponding to 1 mol of the label/mol of the monomeric unit of the enzyme and a decrease of 1 mol of the -SH group. 5) Inactivation was accompanied by the formation of a new absorption peak at 320 nm which was bleached by addition of NaBH4.     

Antimicrobial susceptibility and genetic relatedness of bovine Stenotrophomonas maltophilia isolates from a mastitis outbreak

Aims: To evaluate the antimicrobial susceptibility and genetic relatedness of 11 Stenotrophomonas maltophilia isolates from an outbreak of bovine clinical mastitis in one herd and two isolates from two separate mastitis cases in two other herds. Methods and Results: Thirteen S. maltophilia isolates were obtained from milk samples from 11 cows from three dairy herds in Japan during 2008. We tested their susceptibility to 14 antimicrobials by broth microdilution and identified their genotypes by enterobacterial repetitive intergenic consensus 2 (ERIC2)‐PCR. Every cow had acute mild mastitis (slightly watery foremilk with flakes) without systemic symptoms and all resolved within 3–5 weeks of diagnosis. Eleven of the 13 isolates derived from nine cows in one herd over a 7‐month period exhibited a closely related ERIC2 type (A). The remaining two isolates derived from two cows from two other herds exhibited two distinct ERIC2 types (B and C). Most of the 13 isolates exhibited susceptibility to trimethoprim‐sulfamethoxazole, chloramphenicol, minocycline and levofloxacin; however, they were resistant to four β‐lactams, kanamycin, gentamicin and oxytetracycline. They were intermediate to enrofloxacin. Conclusions: Eleven closely related S. maltophilia isolates were involved in a herd outbreak of mastitis to some extent. Bovine S. maltophilia isolates exhibited resistance to many classes of antimicrobials. Significance and impact of study: This is a rare report of a herd outbreak of bovine mastitis involving closely related S. maltophilia isolates.     

Changes in sleep quality of athletes under normobaric hypoxia equivalent to 2,000-m altitude: a polysomnographic study.

This study evaluated the sleep quality of athletes in normobaric hypoxia at a simulated altitude of 2,000 m. Eight male athletes slept in normoxic condition (NC) and hypoxic conditions equivalent to those at 2,000-m altitude (HC). Polysomnographic recordings of sleep included the electroencephalogram (EEG), electrooculogram, chin surface electromyogram, and electrocardiogram. Thoracic and abdominal motion, nasal and oral airflow, and arterial blood oxygen saturation (Sa(O(2))) were also recorded. Standard visual sleep stage scoring and fast Fourier transformation analyses of the EEG were performed on 30-s epochs. Subjective sleepiness and urinary catecholamines were also monitored. Mean Sa(O(2)) decreased and respiratory disturbances increased with HC. The increase in respiratory disturbances was significant, but the increase was small and subclinical. The duration of slow-wave sleep (stage 3 and 4) and total delta power (<3 Hz) of the all-night non-rapid eye movement sleep EEG decreased for HC compared with NC. Subjective sleepiness and amounts of urinary catecholamines did not differ between the conditions. These results indicate that acute exposure to normobaric hypoxia equivalent to that at 2,000-m altitude decreased slow-wave sleep in athletes, but it did not change subjective sleepiness or amounts of urinary catecholamines.     

Identification and characterization of XPC-binding domain of hHR23B.

hHR23B was originally isolated as a component of a protein complex that specifically complements nucleotide excision repair (NER) defects of xeroderma pigmentosum group C cell extracts in vitro and was identified as one of two human homologs of the Saccharomyces cerevisiae NER gene product Rad23. Recombinant hHR23B has previously been shown to significantly stimulate the NER activity of recombinant human XPC protein (rhXPC). In this study we identify and functionally characterize the XPC-binding domain of hHR23B protein. We prepared various internal as well as terminal deletion products of hHR23B protein in a His-tagged form and examined their binding with rhXPC by using nickel-chelating Sepharose. We demonstrate that a domain covering 56 amino acids of hHR23B is required for binding to rhXPC as well as for stimulation of in vitro NER reactions. Interestingly, a small polypeptide corresponding to the XPC-binding domain is sufficient to exert stimulation of XPC NER activity. Comparison with known crystal structures and analysis with secondary structure programs provided strong indications that the binding domain has a predominantly amphipathic alpha-helical character, consistent with evidence that the affinity with XPC is based on hydrophobic interactions. Our work shows that binding to XPC alone is required and sufficient for the role of hHR23B in in vitro NER but does not rule out the possibility that the protein has additional functions in vivo.