Design and evaluation of specific PCR primers for rapid and reliable identification of Staphylococcus xylosus strains isolated from dry fermented sausages

Rapid and reliable identification of Staphylococcus xylosus was achieved by species-specific PCR assays. Two sets of primers, targeting on xylulokinase (xylB) and 60 kDa heat-shock protein (hsp60) genes of S. xylosus, respectively, were designed. Species-specificity of both sets of primers was evaluated by using 27 reference strains of the DSM collection, representing 23 different species of the Staphylococcus genus and 3 species of the Kocuria genus. Moreover, 90 wild strains isolated from different fermented dry sausages were included in the analysis. By using primers xylB-F and xylB-R the expected PCR fragment was obtained only when DNA from S. xylosus was used. By contrast, amplification performed by using primers xylHs-F and xylHs-R produced a single PCR fragment, of the expected length, when DNA from S. xylosus, S. haemolyticus, S. intermedius and S. kloosii were used as template. Nevertheless, AluI digestion of the xylHs-F/xylHs-R PCR fragment allowed a clear differentiation of these 4 species. The rapidity (about 4 h from DNA isolation to results) and reliability of the PCR procedures established suggests that the method may be profitably applied for specific detection and identification of S. xylosus strains.     

Human DNA polymerase lambda possesses terminal deoxyribonucleotidyl transferase activity and can elongate RNA primers: implications for novel functions

DNA polymerase lambda is a novel enzyme of the family X of DNA polymerases. The recent demonstration of an intrinsic 5'-deoxyribose-5'-phosphate lyase activity, a template/primer dependent polymerase activity, a distributive manner of DNA synthesis and sequence similarity to DNA polymerase beta suggested a novel beta-like enzyme. All these properties support a role of DNA polymerase lambda in base excision repair. On the other hand, the biochemical properties of the polymerisation activity of DNA polymerase lambda are still largely unknown. Here we give evidence that human DNA polymerase lambda has an intrinsic terminal deoxyribonucleotidyl transferase activity that preferentially adds pyrimidines onto 3'OH ends of DNA oligonucleotides. Furthermore, human DNA polymerase lambda efficiently elongates an RNA primer hybridized to a DNA template. These two novel properties of human DNA polymerase lambda might suggest additional roles for this enzyme in DNA replication and repair processes.     

Quinolinic acid but not MK-801 protects the dopaminergic system from 1-methyl-4- phenyl-1,2,3,6-tetrahydropyridine-induced toxicity in goldfish retina

The toxicity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, intravitreally injected in goldfish eye, involves interplexiform retinal neurons and depletes tyrosine hydroxylase immunoreactivity and dopamine levels. This induced neurotoxicity was prevented by the concomitant administration in nontoxic doses (10 μg) of quinolinic acid, an endogenous structural analogue of N-methyl -aspartate with excitotoxic properties. Quinolinic acid is ineffective on the retinal degeneration induced by 1-methyl-4- phenylpyridinium ion. This fact suggests that quinolinic acid inhibits the MAO-B oxidation of 1-methyl-4- phenyl-1,2,3,6-tetrahydropyridine. MK-801, a noncompetitive antagonist of glutamate NMDA-receptors, exerts partial protective effects on MPTP-induced delayed toxicity in mammals. In the goldfish eye, MK-801, injected in low concentration, and in conjunction with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine or 1-methyl-4-phenylpyridinium ion, did not prevent retinal neurodegeneration. Ten μg of MK-801 alone did not affect retinal neurons, while a higher concentration (20 μg) causes the chromatolysis of some photoreceptor nuclei.     

Comparative mapping between<Emphasis Type="Italic"> Quercus</Emphasis> and<Emphasis Type="Italic"> Castanea</Emphasis> using simple-sequence repeats (SSRs)

Simple sequence repeat (SSR) markers from Quercus and Castanea were used for comparative mapping between Quercus robur (L.) and Castanea sativa (Mill.). We tested the transferability of SSRs developed in Quercus to Castanea and vice-versa. In total, 47% (25) of the Quercus SSRs and 63% (19) of the Castanea SSRs showed a strong amplification product in the non-source species. From these 44 putative comparative anchor tags, 19 (15 from Quercus and 4 from Castanea) were integrated in two previously established genetic linkage maps for the two genera. SSR loci were sequenced to confirm the orthology of the markers. The combined information from both genetic mapping and sequence analysis were used to determine the homeology between seven linkage groups, aligned on the basis of pairs or triplets of common markers, while two additional groups were matched using a single microsatellite marker. Orthologous loci identified between Q. robur and C. sativa will be useful as anchor loci for comparative mapping studies within the Fagaceae family.Communicated by D.B. NealeThis paper is dedicated to the memory of Paulo Costa     

Antimicrobial activity of a nisin-activated plastic film for food packaging

AIMS: To determine the effectiveness of a packaging film coated with nisin to inhibit Micrococcus luteus ATCC 10240 in tryptone soya broth (TSB) and the microbiota of raw milk during storage. A further aim was to examine the release of nisin from the activated film. METHODS AND RESULTS: An active package, obtained from nisin-treated film, was filled with 1 l of M. luteus ATCC 10240 (ML) suspension in TSB and stored at 4 and 25 degrees C for 2 days. After 24 h at 25 degrees C there was a remarkable reduction of M. luteus ATCC 10240 compared with the control, while at 4 degrees C a slight reduction was observed. Moreover, microbial growth was controlled when 1 l of three different kinds of milk was poured into the active package and stored at 4 degrees C for 7 days. The most significant results were observed in raw milk and pasteurized milk with a reduction of 0.9 log and 1.3 log, respectively. The release experiments showed that nisin release from the film was unpredictable, but it was favoured by low pH and high temperature. CONCLUSIONS: It appears that nisin-coated films were effective in inhibiting M. luteus ATCC 10240 in TSB and the bacterial flora in milk, and the release of nisin was pH and temperature dependent. SIGNIFICANCE AND IMPACT OF THE STUDY: Nisin-activated film may control bacterial growth, maintaining food quality, safety and extending the shelf-life of food products.     

Preliminary studies on levels of sensitivity to phosphoric esters in the Italian population of the Culex pipiens complex

A preliminary survey of the susceptibility level to organophosphate (OP) insecticides in six populations of Culex pipiens from Central Italy has been carried out. The correlation between OP resistance and highly active esterase electromorphs has also been investigated. The bioassay and electrophoresis results show an association between OP resistance and the highly active esterase allele Est-3A and confirm what it was found in populations of C. pipiens from Southern France. Est-3A appears to be a reliable indicator of OP resistance in mosquitoes of italian C. pipiens.     

Statistical and ecological analysis for the evaluation of floristic diversity: The case of a volcanic complex in North-eastern Italy (Colli Euganei,Padova, Italy)

ABSTRACT

The Euganean Hills are a volcanic mountainous complex, which rises from the eastern Po Plain, and consists of about one hundred hills, rising from 80 m to 604 m above sea level. In the studied area, 1158 taxa of Tracheophytes have been collected. The chorological and life-form analyses of this flora have shown the predominance of the Mediterranean s.l. elements and of the hemicryptophytes, followed by the therophytes. The chorological aspect of this floristic complex was analysed. A cross-spectrum was calculated to see how life-forms are represented in the different chorotypes. A comparison with the biological and chorological spectra of the Veneto Region and of Italy as a whole was also carried out. Several species of remarkable floristic and phytogeographical interest have been found, as well as some included in the national and Regional lists of protected species.     

DNA polymerase alpha, beta and gamma activities in rabbit spleen cell populations stimulated by various doses of concanavalin A

Adherence and phagocytosis of ⁵¹chromium labeled sheep red blood cells ([⁵¹Cr]-sRBC) by P388 D₁ cells in tissue culture were studied under various conditions and were found to possess certain requirements including opsonization, temperature, microfilaments and cyclic nucleotide levels. Exogenous administration of 10^?2 M N⁶, O²-dibutyryl adenosine 3′–5′ cyclic monophosphoric acid (db-cAMP) or adenosine 3′–5′ cyclic monophosphoric acid (cAMP) inhibited phagocytosis of opsonized [⁵¹Cr]-sRBC by 36 and 42%, respectively. Aminophylline potentiated the inhibitory response to both cAMP and db-cAMP. The measurement of endogenous cyclic nucleotide levels during phagocytosis of opsonized sRBC showed a rise in guanosine 3′–5′ cyclic monophosphate (cGMP) during the first 5 min with a gradual decline to control levels at 45 min and a rise in cAMP levels reaching a peak at 30 min which remained above control values for the duration of the experiment. As the rate of phagocytosis decreased the ratio of $\text{cAMP}\text{cGMP}$ increased. These observations emphasize the importance of metabolic functions and cyclic nucleotides during phagocytosis by the P388 D₁ cells and strengthen the usefulness of the P388 D₁ cells as a model in evaluating various macrophage activities.     

Restrictions to carbon dioxide conductance and photosynthesis in spinach leaves recovering from salt stress

Salt accumulation in spinach (Spinacia oleracea L.) leaves first inhibits photosynthesis by decreasing stomatal and mesophyll conductances to CO₂ diffusion and then impairs ribulose-1,5-bisphosphate carboxylase/oxygenase (S. Delfine, A. Alvino, M. Zacchini, F. Loreto [1998] Aust J Plant Physiol 25: 395–402). We measured gas exchange and fluorescence in spinach recovering from salt accumulation. When a 21-d salt accumulation was reversed by 2 weeks of salt-free irrigation (rewatering), stomatal and mesophyll conductances and photosynthesis partially recovered. For the first time, to our knowledge, it is shown that a reduction of mesophyll conductance can be reversed and that this may influence photosynthesis. Photosynthesis and conductances did not recover when salt drainage was restricted and Na content in the leaves was greater than 3% of the dry matter. Incomplete recovery of photosynthesis in rewatered and control leaves may be attributed to an age-related reduction of conductances. Biochemical properties were not affected by the 21-d salt accumulation. However, ribulose-1,5-bisphosphate carboxylase/oxygenase activity and content were reduced by a 36- to 50-d salt accumulation. Photochemical efficiency was reduced only in 50-d salt-stressed leaves because of a decrease in the fraction of open photosystem II centers. A reduction in chlorophyll content and an increase in the chlorophyll a/b ratio were observed in 43- and 50-d salt-stressed leaves. Low chlorophyll affects light absorptance but is unlikely to change light partitioning between photosystems.     

Genetic Variation in the Familial Mediterranean Fever Gene (MEFV) and Risk for Crohn's Disease and Ulcerative Colitis

Background and Aims

The familial Mediterranean fever (FMF) gene (MEFV) encodes pyrin, a major regulator of the inflammasome platform controlling caspase-1 activation and IL-1β processing. Pyrin has been shown to interact with the gene product of NLRP3, NALP3/cryopyrin, also an important active member of the inflammasome. The NLRP3 region was recently reported to be associated with Crohn''s disease (CD) susceptibility. We therefore sought to evaluate MEFV as an inflammatory bowel disease (IBD) susceptibility gene.

Methodology and Results

MEFV colonic mucosal gene expression was significantly increased in experimental colitis mice models (TNBS p<0.0003; DSS p<0.006), in biopsies from CD (p<0.02) and severe ulcerative colitis (UC) patients (p<0.008). Comprehensive genetic screening of the MEFV region in the Belgian exploratory sample set (440 CD trios, 137 UC trios, 239 CD cases, 96 UC cases, and 107 healthy controls) identified SNPs located in the MEFV 5′ haplotype block that were significantly associated with UC (rs224217; p = 0.003; A allele frequency: 56% cases, 45% controls), while no CD associations were observed. Sequencing and subsequent genotyping of variants located in this associated haplotype block identified three synonymous variants (D102D/rs224225, G138G/rs224224, A165A/rs224223) and one non-synonymous variant (R202Q/rs224222) located in MEFV exon 2 that were significantly associated with UC (rs224222: p = 0.0005; A allele frequency: 32% in cases, 23% in controls). No consistent associations were observed in additional Canadian (256 CD trios, 91 UC trios) and Scottish (495 UC, 370 controls) sample sets. We note that rs224222 showed marginal association (p = 0.012; G allele frequency: 82% in cases, 70% in controls) in the Canadian sample, but with a different risk allele. None of the NLRP3 common variants were associated with UC in the Belgian-Canadian UC samples and no significant interactions were observed between NLRP3 and MEFV that could explain the observed flip-flop of the rs224222 risk allele.

Conclusion

The differences in association levels observed between the sample sets may be a consequence of distinct founder effects or of the relative small sample size of the cohorts evaluated in this study. However, the results suggest that common variants in the MEFV region do not contribute to CD and UC susceptibility.