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.     

Changes in the spoilage-related microbiota of beef during refrigerated storage under different packaging conditions

The microbial spoilage of beef was monitored during storage at 5 degrees C under three different conditions of modified-atmosphere packaging (MAP): (i) air (MAP1), (ii) 60% O2 and 40% CO2 (MAP2), and (iii) 20% O2 and 40% CO2 (MAP3). Pseudomonas, Enterobacteriaceae, Brochothrix thermosphacta, and lactic acid bacteria were monitored by viable counts and PCR-denaturing gradient gel electrophoresis (DGGE) analysis during 14 days of storage. Moreover, headspace gas composition, weight loss, and beef color change were also determined at each sampling time. Overall, MAP2 was shown to have the best protective effect, keeping the microbial loads and color change to acceptable levels in the first 7 days of refrigerated storage. The microbial colonies from the plate counts of each microbial group were identified by PCR-DGGE of the variable V6-V8 region of the 16S rRNA gene. Thirteen different genera and at least 17 different species were identified after sequencing of DGGE fragments that showed a wide diversity of spoilage-related bacteria taking turns during beef storage in the function of the packaging conditions. The countable species for each spoilage-related microbial group were different according to packaging conditions and times of storage. In fact, the DGGE profiles displayed significant changes during time and depending on the initial atmosphere used. The spoilage occurred between 7 and 14 days of storage, and the microbial species found in the spoiled meat varied according to the packaging conditions. Rahnella aquatilis, Rahnella spp., Pseudomonas spp., and Carnobacterium divergens were identified as acting during beef storage in air (MAP1). Pseudomonas spp. and Lactobacillus sakei were found in beef stored under MAP conditions with high oxygen content (MAP2), while Rahnella spp. and L. sakei were the main species found during storage using MAP3. The identification of the spoilage-related microbiota by molecular methods can help in the effective establishment of storage conditions for fresh meat.     

Conversion of monofunctional DNA adducts of cis-diamminedichloroplatinum (II) to bifunctional lesions. Effect on the in vitro replication of single-stranded DNA by Escherichia coli DNA polymerase I and eukaryotic DNA polymerases alpha

Reaction of cis-diamminedichloroplatinum (II) with single-stranded M13 phage DNA in vitro produced monofunctional platinum-DNA adducts on guanine and bifunctional lesions with either two guanine bases (GG) or one adenine and one guanine (AG). When DNA containing a majority of monofunctional platinum-DNA lesions was dialyzed against 10 mM NaCIO4 at 37 degrees C, conversion of monoadducts to bifunctional lesions was observed. We examined the effect of post-treatment formation of bifunctional lesions on DNA synthesis by Escherichia coli DNA polymerase I and highly purified eukaryotic DNA polymerase alpha from Drosophila melanogaster and calf thymus. Arrest sites on the platinated template were determined by polyacrylamide gel electrophoresis. Monofunctional lesions did not appear to block DNA synthesis. Inhibition of replication increased as bifunctional platinum-DNA lesions formed during post-treatment incubation; GG adducts inhibited replication more than AG. These results suggest that bifunctional GG platinum-DNA adducts may be the major toxic damage of cisplatin.     

16S-23S rDNA intergenic spacer region polymorphism of Lactococcus garvieae,Lactococcus raffinolactis and Lactococcus lactis as revealed by PCR and nucleotide sequence analysis

The intergenic spacer region (ISR) between the 16S and 23S rRNA genes was tested as a tool for differentiating lactococci commonly isolated in a dairy environment. 17 reference strains, representing 11 different species belonging to the genera Lactococcus, Streptococcus, Lactobacillus, Enterococcus and Leuconostoc, and 127 wild streptococcal strains isolated during the whole fermentation process of "Fior di Latte" cheese were analyzed. After 16S-23S rDNA ISR amplification by PCR, species or genus-specific patterns were obtained for most of the reference strains tested. Moreover, results obtained after nucleotide analysis show that the 16S-23S rDNA ISR sequences vary greatly, in size and sequence, among Lactococcus garvieae, Lactococcus raffinolactis, Lactococcus lactis as well as other streptococci from dairy environments. Because of the high degree of inter-specific polymorphism observed, 16S-23S rDNA ISR can be considered a good potential target for selecting species-specific molecular assays, such as PCR primer or probes, for a rapid and extremely reliable differentiation of dairy lactococcal isolates.     

Micronucleated lymphocyte rates from head-and-neck cancer patients

We investigated whether head-and-neck cancers are associated with an increased micronucleated cell rates (MN cell rates) and whether risk factors for these cancers are associated with alterations in micronucleated lymphocytes. MN cell rates were assessed in cytokinesis-blocked lymphocytes of 57 head-and-neck cancer patients (CP) before any anticancer treatment and of 198 male and female healthy subjects (HS). In the HS group, only smoking status significantly affect MN cell rates. In CP group age, sex, tobacco status, alcohol status, tumor stage, family history of cancer had no significant effect. For the non-smokers, the comparison between MN cell rates in HS and CP adjusted for age and sex showed a significant difference. The increase of MN cell rates in non-smokers patients may be attributable to cancer status. For the smokers, the comparison of MN cell rates in HS and CP matched for age and sex showed no significant difference. Pathological status could mask the smoking effect on peripheral blood lymphocytes in patients. Moreover, it probably could partly explain why MN cell rates in matched-CP smokers and HS smokers were similar. The authors do not recommend the CBMN assay in this present form to study smoking DNA-damage effects in peripheral blood lymphocytes of cancer patients, especially for patients with upper aero-digestive tract cancers or lung cancers for which tobacco is the major risk factor.     

Ab Initio Studies on N-Methylacetamide

The potential energy surface of N-methylacetamide (NMA) is investigated ab initio at the Hartree–Fock/6-31G* level with full geometry optimisation with or without MP2 correlation and thermal corrections in order to characterise its stationary points and the Intrinsic Reaction Coordinate (IRC) of trans-cis isomerisation. The cis and trans minima and the two non-equivalent Transition States (TS) of the isomerisation with opposite pyramidalisation of the amidic group are determined. The TS result considerably destablised (16 and 21 kcal mol-1) with respect to the most stable equilibrium structure. The lowest energy transition state is the up form (both substituents at N bent toward the carbonyl oxygen), while the down form (with the N lone pair pointing toward the carbonyl oxygen) is higher in energy. A saddle point of index 2 connects the two transition states, 8 kcal mol-1 above the TS up, the absence of intermediates being verified. Adding the correlation corrections does not change the energy gaps appreciably. The IRC at HF/6-31G* level has been followed in the most favourable case: correlate deformation of peptide unit and decoupling of methyl groups with respect to the reaction coordinate are shown. Additional TS, depending on methyl groups orientation, confirm the substantial free rotation around the phiv; and torsional angles.     

Tissue-specific changes of mitochondrial functions in aged rats: effect of a long-term dietary treatment with N-acetylcysteine

The understanding of the involvement of mitochondrial oxidative phosphorylation (OXPHOS) in the aging process has often been biased by the different methodological approaches as well as the choice of the biological material utilized by the various groups. In the present paper, we have carried out a detailed analysis of several bioenergetic parameters and oxidative markers in brain and heart mitochondria from young (2 months) and old (28 months) rats. This analysis has revealed an age-related decrease in respiratory fluxes in brain but not in heart mitochondria. The age-related decrease in respiratory rate (-43%) by NAD-dependent substrates was associated with a consistent decline (-40%) of complex I activity in brain mitochondria. On the other hand, heart mitochondria showed an age-related decline of complex II activity. Both tissues showed, however, an age-associated accumulation of oxidative damage. We have then performed the same analysis on old (28 months) rats subjected to a long-term (16 months) diet containing the antioxidant N-acetylcysteine (NAC). The treated old rats showed a slight brain-specific improvement of mitochondrial energy production efficiency, mostly with NAD-dependent substrates, together with a decrease in carbonyl protein content and an increase in the amount of protein thiols of brain cytosolic fraction. A full recovery of complex II activity was detected in heart mitochondria from NAC-treated old rats. The present work documents the marked tissue specificity of the decline of bioenergetic functions in isolated mitochondria from aged rats and provides the first data on the effects of a long-term treatment with N-acetylcysteine.     

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.