Heat shock and thermotolerance of Escherichia coli O157:H7 in a model beef gravy system and ground beef.

Duplicate beef gravy or ground beef samples inoculated with a suspension of a four-strain cocktail of Escherichia coli O157:H7 were subjected to sublethal heating at 46 degrees C for 15-30 min, and then heated to a final internal temperature of 60 degrees C. Survivor curves were fitted using a linear model that incorporated a lag period (TL), and D-values and 'time to a 4D inactivation' (T4D) were calculated. Heat-shocking allowed the organism to survive longer than non-heat-shocked cells; the T4D values at 60 degrees C increased 1.56- and 1.50-fold in beef gravy and ground beef, respectively. In ground beef stored at 4 degrees C, thermotolerance was lost after storage for 14 h. However, heat-shocked cells appeared to maintain their thermotolerance for at least 24 h in ground beef held to 15 or 28 degrees C. A 25 min heat shock at 46 degrees C in beef gravy resulted in an increase in the levels of two proteins with apparent molecular masses of 60 and 69 kDa. These two proteins were shown to be immunologically related to GroEL and DnaK, respectively. Increased heat resistance due to heat shock must be considered while designing thermal processes to assure the microbiological safety of thermally processed foods.

Sensitive detection of viable Listeria monocytogenes by reverse transcription-PCR.

Detection of pathogens in contaminated food products by PCR can result in false-positive data due to the amplification of DNA from nonviable cells. A new method based on reverse transcription-PCR (RT-PCR) amplification of mRNA for the specific detection of viable Listeria monocytogenes was developed. The expression of three L. monocytogenes genes, iap, hly, and prfA, was examined to determine a suitable target for amplification of RT-PCR. Total RNA from L. monocytogenes was isolated, and following DNase treatment, the RNA was amplified by both RT-PCR and PCR with primers specific for the three genes. Amplicon detection was accomplished by Southern hybridization to digoxigenin-labeled gene probes. The levels of expression of these three genes differed markedly, and the results indicated that the iap gene would provide a good target for development of a specific method for detection of viable L. monocytogenes based on RT-PCR amplification. After a 1-h enrichment, the 371-bp iap-specific product was detected with a sensitivity of ca. 10 to 15 CFU/ml from pure culture. Detection of the 713-bp hly-specific amplicon was ca. 4,000 times less sensitive after 1 h, whereas detection of the 508-bp prfA product showed the lowest level of sensitivity, with detection not observed until after a 5-h enrichment period. The amplification of the iap mRNA was specific for L. monocytogenes. Overall, the assay could be completed in ca. 54 h. The use of RT-PCR amplification for the detection of viable L. monocytogenes was validated in artificially contaminated cooked ground beef. Following a 2-h enrichment incubation, the iap-specific amplification product could be detected in a cooked meat sample that was originally inoculated with ca. 3 CFU/g. These results support the usefulness of RT-PCR amplification of mRNA as a sensitive method for the specific detection of viable L. monocytogenes and indicate that this method may prove useful in the detection of this pathogen in ready-to-eat, refrigerated meat products.

Early clinical results of the use of radiofrequency lesioning in the treatment of plantar fasciitis.

In this paper the authors discuss plantar fascial heel pain, including its etiology and treatment. The results of a retrospective study using radiofrequency lesioning on plantar fascial heel pain are presented and discussed. Thirty-nine patients were treated with radiofrequency lesioning, and 92% of the patients experienced complete resolution of symptoms following the procedure.

Replacement of the tyrosine residue that links a potyviral VPg to the viral RNA is lethal.

Mutants of tobacco vein mottling virus (TVMV) were constructed in which the tyrosine residue (Tyr1860) that links the VPg to the viral RNA was changed to phenylalanine or serine or was inverted in position with the adjacent glycine residue. In another mutant, the tyrosine residue nearest to Tyr1860 (Tyr1867) was changed to a phenylalanine residue. The resulting mutants were tested for their ability to infect Nicotiana tabacum plants or protoplasts. The Tyr1860 mutants did not accumulate to detectable levels in infected plants when tested by ELISA and Northern blot analysis. Moreover, the Tyr1860-associated mutants were not infectious in protoplasts, indicating that mutations involving the linking amino acid of the TVMV VPg abolished viral replication. In contrast to the Tyr1860 mutants, transcripts from the mutation of Tyr1867 to a phenylalanine residue infected both protoplasts and plants. Analysis of progeny RNA from plants inoculated with the Tyr1867 mutant indicated that a reversion to wild type had occurred in systemically infected leaves.

A potyvirus polymerase interacts with the viral coat protein and VPg in yeast cells.

The two-hybrid system was used to test for pairwise interactions between the tobacco vein mottling virus (TVMV)-encoded RNA-dependent RNA polymerase (or NIb protein) and two other TVMV-encoded proteins: the NIa protein, which consists of genome-linked protein (VPg) and proteinase domains, and the viral coat protein (CP). Using this approach, we find that the NIb protein interacts with both the NIa protein and the CP in yeast cells. Moreover, we find that a mutation in the conserved GDD domain of the NIb protein diminishes the NIb-CP interaction but not the NIb-NIa interaction. Likewise, mutations in the vicinity of the NIa protein to which the genomic RNA is covalently attached eliminate the NIb-NIa interaction. We conclude that the NIb protein interacts with the VPg domain of the NIa protein and that this interaction requires a functional RNA attachment site. This interaction may be important for the initiation of viral RNA synthesis in infected cells. We also conclude that the CP interacts with the NIb in a manner that is sensitive in changes in the highly conserved GDD motif. The role of this interaction in the functioning of the NIb protein or the CP is unclear, but may involve regulation of viral RNA synthesis in infected cells.

Toward tougher oversight of managed care: MSNJ proposals.

The Medical Society of New Jersey has developed a detailed position statement on the regulation of health maintenance organizations (HMO). This statement has been presented to an advisory committee of the New Jersey State Department of Health, which is preparing new HMO regulations.

Mutational analysis of the tobacco vein mottling virus genome.

We have used a cDNA clone of the potyvirus, tobacco vein mottling virus, to construct 19 mutants bearing 12-nt insertions in the viral genome. These mutants display a variety of phenotypes in inoculated tobacco plants or protoplasts. All mutants with insertions in P3, CI, 6K, NIa, or NIb failed to produce detectable amounts of progeny viral RNA in protoplasts or plants which suggests that they all may be directly involved in replication. Mutants (one in P1 and one in HCpro) presumably affected in polyprotein processing also did not replicate in plants or protoplasts. Seven mutants, with insertions in the 5' noncoding region, P1, HCpro, or CP regions of the genome, were able to infect protoplasts. Three of the 7 mutants (1 in the 5' noncoding region and 2 in HCpro) were able to infect protoplasts but not plants. The remaining 4 mutants replicated in protoplasts and were able to cause systemic infection in plants. The mutation in the CP had no effect on virus accumulation or symptom development in inoculated plants, whereas the other 3 (1 in P1 and 2 in HCpro) induced cyclical patterns of symptom expression. These symptoms ranged from very mild to wild-type-like as new leaves emerged and, as the plants continued to grow, this pattern was repeated. These results support the assignment of roles in replication to five coding regions in the genome and demonstrate that sequence alterations in many parts of other regions of the viral genome may have pronounced effects on replication and the expression of disease symptoms.

Synthesis and turnover of photosystem II reaction center protein D1. Ribosome pausing increases during chloroplast development.

The chloroplast photosystem II reaction center protein D1 contains five membrane-spanning helices and binds chlorophyll, carotenoid, quinone, iron, and probably manganese. Turnover of pulse-labeled D1 in isolated plastids was found to involve cleavage between helix IV and helix V, which releases a 23-kDa N-terminal peptide and two C-terminal peptides of 10 and 8 kDa. Ribosomes pause at specific sites during translation of D1, which results in the accumulation of D1 translation intermediates. Pulse-labeling assays followed by polysome isolation and immunoprecipitation identified paused D1 translation intermediates of 9, 12.5, 15-18, 20, 21, 24, and 28-32 kDa. Ribosome pausing was not altered when dark-grown seedlings were illuminated for up to 1 h, even though this treatment stimulated accumulation of chlorophyll and D1. However, illumination of plants for 16-72 h resulted in increased ribosome pausing and the build-up of D1 translation intermediates. We hypothesize that ribosome pausing during synthesis of D1 improves the efficiency of chlorophyll binding of D1 nascent chains and enhances accumulation of D1 in mature chloroplasts, which have reduced rates of chlorophyll biosynthesis.

Vir-115 gene product is required to stabilize D1 translation intermediates in chloroplasts.

The nuclear gene mutant of barley, vir-115, shows a developmentally induced loss of D1 synthesis that results in inactivation of Photosystem II. Translation in plastids isolated from 1 h illuminated vir-115 seedlings is similar to wild type. In wild-type barley, illumination of plants for 16 to 72 h results in increased radiolabel incorporation into the D1 translation intermediates of 15-24 kDa. In contrast, these D1 translation intermediates were not observed in vir-115 plastids isolated from plants illuminated for 16-72 h. In addition, after 72 h of illumination, radiolabel incorporation into D1 was undetectable in vir-115 plastids. The level and distribution of psbA mRNA in membrane-associated polysomes was similar in wild-type and vir-115 mutant plastids isolated from plants illuminated for 16-72 h. Toeprint analysis showed similar levels of translation initiation complexes on psbA mRNA in vir-115 and wild-type plastids. These results indicate that translation initiation and elongation of D1 is not significantly altered in the mutant plastids. Ribosome pausing on psbA mRNA was observed in wild-type and vir-115 mutant plastids. Therefore, the absence of D1 translation intermediates in mutant plastids is not due to a lack of ribosome pausing on psbA mRNA. Based on these results, it is proposed that vir-115 lacks or contains a modified nuclear-encoded gene product which normally stabilizes the D1 translation intermediates. In wild-type plastids, ribosome pausing and stabilization of D1 translation intermediates is proposed to facilitate assembly of cofactors such as chlorophyll with D1 allowing continued D1 synthesis and accumulation in mature chloroplasts.

A determinant of disease symptom severity is located in the 3'-terminal noncoding region of the RNA of a plant virus.

Inoculation of Nicotiana tabacum plants with RNA transcribed in vitro from a variant (pXBS8) of a cloned full-length DNA copy of tobacco vein mottling virus (TVMV) RNA resulted in attenuation of the vein mottling and blotching symptoms typically produced by transcripts of cloned wild-type cDNA (pXBS7). Similar amounts of virus were detected by ELISA (using anti-TVMV coat protein serum) in systemically infected leaves of plants inoculated with pXBS7 or pXBS8 transcripts. pXBS8 was shown to contain a 58-nucleotide segment in the sequence corresponding to the 3'-terminal untranslated region of TVMV RNA that was not present in pXBS7. This segment resulted in the appearance in pXBS8 transcripts of four adjacent direct repeats of a 14-nucleotide sequence, AUAAUUAUAUAUAU, that is present in the 3'-untranslated region of TVMV RNA, with two additional nucleotides (AU) between the first and second repeats. Insertion of restriction fragments containing the segment into pXBS7 and inoculation of plants with transcripts of the chimeric construct (pXBS78) resulted in the attenuated-symptom phenotype and was not accompanied by a reduced accumulation of virus in the plant as determined by ELISA and Northern blot analysis. When the extra nucleotides were removed from the variant clone, symptoms induced by transcripts of the cDNA (pXBS87) resembled those induced by wild-type transcripts. The results indicate that a noncoding region of the genome can have a direct effect on the induction of disease symptoms by an RNA virus.

Ribosomes pause at specific sites during synthesis of membrane-bound chloroplast reaction center protein D1.

Photosynthetic reaction center protein D1 contains five membrane-spanning alpha-helices which form binding sites for pheophytin, chlorophyll, carotenoids, quinone, Fe2+, and probably Mn2+. D1 translation intermediates of 15 to 28 kD were detected when isolated chloroplasts were pulse-labeled with [35S]methionine. The D1 translation intermediates were associated with membrane polysomes and can be chased into full length D1. The sites of translation pausing were determined by mapping the distribution of ribosomes on D1 mRNA using toeprint analysis. Clusters of toeprint signals were generated by D1 mRNA associated with membranes but not by D1 mRNA in nonpolysomal fractions of the soluble phase or phenol-extracted mRNA. The distribution of ribosomes on D1 mRNA determined by toeprint analysis was consistent with D1 translation intermediates observed with pulse-labeling. Ribosome pausing may facilitate co-translational binding of co-factors such as chlorophyll to D1 and aid the integration of D1 into thylakoid membranes.

Chlorophyll regulates accumulation of the plastid-encoded chlorophyll apoproteins CP43 and D1 by increasing apoprotein stability.

Chlorophyll apoprotein accumulation in higher plant chloroplasts is controlled by light-dependent chlorophyll formation. Dark-grown plants lack chlorophyll and chlorophyll apoproteins. However, the plastid genes encoding the chlorophyll apoproteins are transcribed; chlorophyll apoprotein mRNA accumulates and associates with polysomes in plastids of dark-grown plants. Pulse-labeling assays revealed a population of short-lived proteins in plastids of dark-grown plants. One of these transiently labeled proteins was CP43, a chlorophyll apoprotein associated with photosystem II. Pulse-chase assays showed that newly synthesized CP43 was rapidly degraded in plastids of dark-grown plants, which lack chlorophyll. In contrast, CP43 synthesized in plastids from illuminated plants was stable. The synthesis of D1, a chlorophyll apoprotein of the photosystem II reaction center, was also analyzed in plastids of dark-grown and illuminated plants. Radiolabel accumulation into full-length D1 was only detected in plastids of illuminated plants. However, D1 translation intermediates of 15-25 kDa were detected in both plastid populations. Pulse-chase assays showed that the 15- to 25-kDa D1 translation products were precursors of mature D1 in plastids of illuminated plants. In contrast, in plastids of dark-grown plants, the 15- to 25-kDa translation intermediates were converted into a 23-kDa polypeptide previously suggested to be a proteolytic product of D1. These results indicate that chlorophyll produced in illuminated plants stabilizes D1 nascent polypeptides, which allows accumulation of mature D1.