Genome-scale metabolic model for Lactococcus lactis MG1363 and its application to the analysis of flavor formation.

Lactococcus lactis subsp. cremoris MG1363 is a paradigm strain for lactococci used in industrial dairy fermentations. However, despite of its importance for process development, no genome-scale metabolic model has been reported thus far. Moreover, current models for other lactococci only focus on growth and sugar degradation. A metabolic model that includes nitrogen metabolism and flavor-forming pathways is instrumental for the understanding and designing new industrial applications of these lactic acid bacteria. A genome-scale, constraint-based model of the metabolism and transport in L. lactis MG1363, accounting for 518 genes, 754 reactions, and 650 metabolites, was developed and experimentally validated. Fifty-nine reactions are directly or indirectly involved in flavor formation. Flux Balance Analysis and Flux Variability Analysis were used to investigate flux distributions within the whole metabolic network. Anaerobic carbon-limited continuous cultures were used for estimating the energetic parameters. A thorough model-driven analysis showing a highly flexible nitrogen metabolism, e.g., branched-chain amino acid catabolism which coupled with the redox balance, is pivotal for the prediction of the formation of different flavor compounds. Furthermore, the model predicted the formation of volatile sulfur compounds as a result of the fermentation. These products were subsequently identified in the experimental fermentations carried out. Thus, the genome-scale metabolic model couples the carbon and nitrogen metabolism in L. lactis MG1363 with complete known catabolic pathways leading to flavor formation. The model provided valuable insights into the metabolic networks underlying flavor formation and has the potential to contribute to new developments in dairy industries and cheese-flavor research.

Creation and repair of specific DNA double-strand breaks in vivo following infection with adenovirus vectors expressing Saccharomyces cerevisiae HO endonuclease.

To study DNA double-strand break (DSB) repair in mammalian cells, the Saccharomyces cerevisiae HO endonuclease gene, or its recognition site, was cloned into the adenovirus E3 or E1 regions. Analysis of DNA from human A549 cells coinfected with the E3::HO gene and site viruses showed that HO endonuclease was active and that broken viral genomes were detectable 12 h postinfection, increasing with time up to approximately 30% of the available HO site genomes. Leftward fragments of approximately 30 kbp, which contain the packaging signal, but not rightward fragments of approximately 6 kbp, were incorporated into virions, suggesting that broken genomes were not held together tightly after cleavage. There was no evidence for DSB repair in E3::HO virus coinfections. In contrast, such evidence was obtained in E1::HO virus coinfections of nonpermissive cells, suggesting that adenovirus proteins expressed in the permissive E3::HO coinfection can inhibit mammalian DSB repair. To test the inhibitory role of E4 proteins, known to suppress genome concatemer formation late in infection (Weiden and Ginsberg, 1994), A549 cells were coinfected with E3::HO viruses lacking the E4 region. The results strongly suggest that the E4 protein(s) inhibits DSB repair.

Popliteal-to-distal bypass grafts for limb salvage.

From July 1989 to July 1994, a total of 44 popliteal-to-distal artery bypasses were performed in 36 patients (29 men and seven women, mean age 62 +/- 10 years). These procedures accounted for 8.8% of all infrainguinal revascularizations performed during that period. Risk factors included diabetes in 33 patients (92%), smoking in 18 (50%), and coronary artery disease in 15 (42%). Prior to revascularization all patients were at risk of limb loss. Tissue necrosis was present in 31 cases (71%), ulceration in eight cases (18%), and rest pain in five cases (11%). Patency of the femoral and popliteal arteries was confirmed prior to surgery in all cases. Intraoperative percutaneous angioplasty of the superficial femoral artery was performed in three cases. Proximal anastomosis was made to the distal popliteal artery in all cases. A total of 52 distal anastomoses (eight sequential bypasses) were made on the following arteries: posterior tibial artery in 13 cases, anterior tibial artery in eight cases, peroneal artery in six cases, plantar artery in two cases, and dorsalis pedis artery in 21 cases. The greater saphenous vein was used as graft material in 42 cases (95%) and the lesser saphenous vein in two cases (5%). No patient died during hospitalization. Early bypass occlusion occurred in three cases (6.8%) and led to amputation in all cases. Secondary patency and limb salvage rates at 3 years calculated using the actuarial method were 74% and 82% respectively. Bypass thrombosis due to superficial femoral artery deterioration was not observed in any case. The present study indicates that popliteal-to-distal artery bypass is a simple, durable, and low-risk method of lower limb revascularization. Medium-term results are promising and support routine use of popliteal-to-distal artery bypass for limb salvage in patients without significant stenosis of the superficial femoral artery.

Extension of native aortic valve endocarditis: surgical considerations.

Among 101 consecutive patients operated on for native infective aortic valve endocarditis (53 males, 48 females, mean age 39 years), 69 presented various forms of infectious extension to the surrounding areas. Twenty-six lesions were noted in the aortic roots: 18 annular abscesses, one abscess of the Valsalva sinus and seven aortic wall destructions. Among the subaortic valve pathology, 27 cases of septal lesions were noted and in one case the mitral fibrous trigone was involved. The mitral apparatus was infected in 26 cases, the tricuspid valvule in one case. Both tricuspid and mitral valvular replacements had to be performed in five cases. Among the 16 postoperative atrioventricular blocks, 14 needed a pacemaker. The most frequent causative microorganisms were Staphylococcus aureus and Streptococcus. Surgical management of the lesions consisted of extensive debridement followed by either simple repair of defects or complex reconstructions involving pericardial or synthetic patches or other more complex operations. Early and late mortality rates were 8.5% and 16%; early and late reoperation rates were 6% and 9.5%, respectively. The mean follow-up time was 148 months (12-265 months) with a survival rate of 74% (SE: +/- 0.08) at 10 years. We conclude that, although surgical correction of infective endocarditis may need a complex approach, it provides good results with an acceptable surgical risk.

A modified single-strand annealing model best explains the joining of DNA double-strand breaks mammalian cells and cell extracts.

The joining of DNA double-strand breaks in vivo is frequently accompanied by the loss of a few nucleotides at the junction between the interacting partners. In vitro systems mimic this loss and, on detailed analysis, have suggested two models for the mechanism of end-joining. One invokes the use of extensive homologous side-by-side alignment of the partners prior to joining, while the other proposes the use of small regions of homology located at or near the terminus of the interacting molecules. to discriminate between these two models, assays were conducted both in vitro and in vivo with specially designed substrates. In vitro, molecules with limited terminal homology were capable of joining, but analysis of the junctions suggested that the mechanism employed the limited homology available. In vivo, the substrates with no extensive homology end-joined with equal efficiency to those with extensive homology in two different topological arrangements. Taken together, these results suggest that extensive homology is not a prerequisite for efficient end-joining, but that small homologies close to the terminus are used preferentially, as predicted by the modified single-strand annealing model.

Characterization of DNA end joining in a mammalian cell nuclear extract: junction formation is accompanied by nucleotide loss, which is limited and uniform but not site specific.

Mammalian cells have a marked capacity to repair double-strand breaks in DNA, but the molecular and biochemical mechanisms underlying this process are largely unknown. A previous report has described an activity from mammalian cell nuclei that is capable of multimerizing blunt-ended DNA substrates (R. Fishel, M.K. Derbyshire, S.P. Moore, and C.S.H. Young, Biochimie 73:257-267, 1991). In this report, we show that nuclear extracts from HeLa cells contain activities which preferentially join linear plasmid substrates in either a head-to-head or tail-to-tail configuration, that the joining reaction is covalent, and that the joining is accompanied by loss of sequence at the junction. Sequencing revealed that there was a loss of a uniform number of nucleotides from junctions formed from any one type of substrate. The loss was not determined by any simple site-specific mechanism, but the number of nucleotides lost was affected by the precise terminal sequence. There was no major effect on the efficiency or outcome of the joining reaction with substrates containing blunt ends or 3' or 5' protruding ends. Using a pair of plasmid molecules with distinguishable restriction enzyme sites, we also observed that blunt-ended DNA substrates could join with those containing protruding 3' ends. As with the junctions formed between molecules with identical ends, there was uniform loss of nucleotides. Taken together, the data are consistent with two models for the joining reaction in which molecules are aligned either throughout most of their length or by using small sequence homologies located toward their ends. Although either model can explain the preferential formation of head-to-head and tail-to-tail products, the latter predicts the precise lossof nucleotides observed. These activities are found in all cell lines examined so far and most likely represent an important repair activity of the mammalian cell.