Early Stages of Conjugation in Escherichia coli

We initiated these studies to learn more about the initial events during bacterial conjugation and to optimize conditions for their occurrence. We found that cells in donor cultures grown anaerobically prior to mating have (i) a higher mean number of F pili per cell, (ii) longer F pili, (iii) a higher probability of forming specific pairs with F(-) cells, and (iv) a faster rate of initiation of chromosome transfer than cells grown aerobically. The growth medium for the donor culture also influences these same parameters: a rich medium is superior to a completely synthetic medium. Starvation of donor cells in buffered saline or for a required amino acid results in (i) a loss of F pili, (ii) a loss in the ability of donor-specific phages to adsorb, (iii) a loss of ability to form specific pairs with F(-) cells and to yield recombinants, and (iv) an increase in recipient ability. These changes occur as a function of starvation time, and at rates which are dependent on the conditions of prior growth and starvation of the donor culture. Either treatment provides a rapid method for the production of F(-) phenocopies from donor cultures. Resynthesis of F pili by cells within a starved donor culture commences very soon after restoration of normal growth conditions, but full restoration of donor ability, as measured by recombinant yield, occurs at a slower rate. We found, along with other investigators, that F pili are essential for specific pair formation. We also found, however, that the presence of F pili is not sufficient for display of donor ability, nor is the absence of F pili enough for cells to exhibit recipient ability. This suggests, therefore, that one or more components, in addition to F pili, are necessary for the conversion of specific pairs to effective pairs (or for chromosome mobilization, or both) and for preventing donor cells from acting as recipients. On the basis of our results, we suggest optimal conditions for achieving high mating efficiencies.     

A comparative study on two fungal lipases from Thermomyces lanuginosus and Yarrowia lipolytica shows the combined effects of detergents and pH on lipase adsorption and activity

The effects of various detergents and pH on the interfacial binding and activity of two fungal lipases from Yarrowia lipolytica (YLLIP2) and Thermomyces lanuginosus (TLL) were investigated using trioctanoin emulsions as well as monomolecular films spread at the air-water interface. Contrary to TLL, YLLIP2 was found to be more sensitive than TLL to interfacial denaturation but it was protected by detergent monomers and lowering the temperature. At pH 7.0, both the interfacial binding and the activities on trioctanoin of YLLIP2 and TLL were inhibited by sodium taurodeoxycholate (NaTDC). At pH 6.0, however, YLLIP2 remained active on trioctanoin in the presence of NaTDC, whereas TLL did not. YLLIP2 activity on trioctanoin was associated with strong interfacial binding of the enzyme to trioctanoin emulsion, whereas TLL was mostly detected in the water phase. The combined effects of bile salts and pH on lipase activity were therefore enzyme-dependent. YLLIP2 binds more strongly than TLL at oil-water interfaces at low pH when detergents are present. These findings are particularly important for lipase applications, in particular for enzyme replacement therapy in patients with pancreatic enzyme insufficiency since high detergent concentrations and highly variable pH values can be encountered in the GI tract.     

Immunocytochemical localization of scorpion digestive lipase

The scorpion hepatopancreas consists of digestive diverticula and interstitial tissue. A digestive diverticulum is composed of two differentiated cell types: the secretory zymogene-like cells and the digestive cells which are the most abundant. The scorpion digestive lipase (SDL) has been previously purified from scorpion hepatopancreas, but its cellular localization has not yet been established. Polyclonal antibodies specific to SDL were prepared and used in immunofluorescence and immunogold techniques to determine the cellular location of SDL. Our results clearly established that SDL was detected intracellularly in specific vesicles tentatively named (SDL+) granules of the digestive cells. No immunolabelling was observed in secretory zymogene-like cells. This immunocytolocalization indicates that lipid digestion might occur in specific granules inside the digestive cells, as suggested by previous studies on the scorpion digestive process.     

S-adenosyl methionine protects ob/ob mice from CYP2E1-mediated liver injury

Pyrazole treatment to induce cytochrome P-450 2E1 (CYP2E1) was recently shown to cause liver injury in ob/ob mice but not in lean mice. The present study investigated the effects of S-adenosyl-l-methionine (SAM) on the CYP2E1-dependent liver injury in ob/ob mice. Pyrazole treatment of ob/ob mice for 2 days caused necrosis, steatosis, and elevated serum transaminase and triglyceride levels compared with saline ob/ob mice. Administration of SAM (50 mg/kg body wt ip every 12 h for 3 days) prevented the observed pathological changes as well as the increase of apoptotic hepatocytes, caspase 3 activity, and serum TNF-alpha levels. SAM administration inhibited CYP2E1 activity but not CYP2E1 content. The pyrazole treatment increased lipid peroxidation, 4-hydroxynonenal and 3-nitrotyrosine protein adducts, and protein carbonyls. These increases in oxidative and nitrosative stress were prevented by SAM. Treatment of ob/ob mice with pyrazole lowered the endogenous SAM levels, and these were elevated after SAM administration. Mitochondrial GSH levels were very low after pyrazole treatment of the ob/ob mice; this was associated with elevated levels of malondialdehyde and 4-hydroxynonenal and 3-nitrotyrosine protein adducts in the mitochondria. All these changes were prevented with SAM administration. SAM protected against pyrazole-induced increase in serum transaminases, necrosis, triglyceride levels, caspase-3 activity, and lipid peroxidation even when administered 1 day after pyrazole treatment. In the absence of pyrazole, SAM lowered the slightly elevated serum transaminases, triglyceride levels, caspase-3 activity, and lipid peroxidation in obese mice. In conclusion, SAM protects against and can also reverse or correct CYP2E1-induced liver damage in ob/ob mice.     

Mapping in the era of sequencing: high density genotyping and its application for mapping TYLCV resistance in Solanum pimpinellifolium

Background

A RIL population between Solanum lycopersicum cv. Moneymaker and S. pimpinellifolium G1.1554 was genotyped with a custom made SNP array. Additionally, a subset of the lines was genotyped by sequencing (GBS).

Results

A total of 1974 polymorphic SNPs were selected to develop a linkage map of 715 unique genetic loci. We generated plots for visualizing the recombination patterns of the population relating physical and genetic positions along the genome.This linkage map was used to identify two QTLs for TYLCV resistance which contained favourable alleles derived from S. pimpinellifolium. Further GBS was used to saturate regions of interest, and the mapping resolution of the two QTLs was improved. The analysis showed highest significance on Chromosome 11 close to the region of 51.3 Mb (qTy-p11) and another on Chromosome 3 near 46.5 Mb (qTy-p3). Furthermore, we explored the population using untargeted metabolic profiling, and the most significant differences between susceptible and resistant plants were mainly associated with sucrose and flavonoid glycosides.

Conclusions

The SNP information obtained from an array allowed a first QTL screening of our RIL population. With additional SNP data of a RILs subset, obtained through GBS, we were able to perform an in silico mapping improvement to further confirm regions associated with our trait of interest. With the combination of different ~ omics platforms we provide valuable insight into the genetics of S. pimpinellifolium-derived TYLCV resistance.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1152) contains supplementary material, which is available to authorized users.     

Genetic and pharmacological inhibition of poly(ADP-ribose) polymerase-1 interferes in the chlamydial life cycle

Chlamydiaceae are intracellular bacteria responsible for a variety of infections, ranging from asymptomatic to very severe, in humans and animals. We have investigated the role of poly(ADP-ribose) polymerase-1 (PARP-1) in Chlamydophila abortus infection using PARP-1-/- and their littermates PARP-1+/+ mice. Infection was resolved more efficiently by PARP-1-/- than PARP-1+/+ mice. However, the inflammatory response was similar in both strains, suggesting a potential role for PARP-1 in the cross-talk between this microorganism and the host cells. PARP-1-/- fibroblasts showed a 10-fold lower rate of chlamydiae production than PARP-1+/+. Moreover, a strong inhibition of bacterial production was also observed after pharmacological inhibition of PARP-1 activity in McCoy cells. Likewise, PARP-1 inhibition induced a higher level of cell death of infected cells, interfering in this way with the normal bacterial cell cycle. Overall, we identify PARP-1 as a new molecule involved in chlamydial developmental cycle, although the intrinsic mechanisms deserve further studies.     

Current knowledge of iron metabolism

Iron plays many roles in human physiology. In this article, we summarize the basic and current knowledge of this essential micronutrient on human metabolism.     

Evaluation of salt tolerance in cultivated and wild tomato species through in vitro shoot apex culture

The possibility of using in vitro shoot apex culture to evaluate salt tolerance of cultivated (Lycopersicon esculentum Mill.) and wild (Lycopersicon pennellii (Correll) D'Arcy) tomato species was determined and related to the response obtained by callus culture. Both apices and calluses were grown on media supplemented with 0, 35, 70, 105, 140, 175 and 210 mM NaCl, and growth and physiological traits were determined. Most apices of L. esculentum did not develop roots from low NaCl levels, whereas the apices of L. pennellii were able to develop roots at the different salt levels. This different degree of salt tolerance between L. esculentum and L. pennellii was not, however, clearly shown on the basis of the shoot growth of the plantlets. The callus response was similar to that shown by the rooting parameters, as callus growth in response to increased salinity was much greater in L. pennellii than in the tomato cultivar. K⁺decreased more and proline accumulated less with salinity in shoots of L. esculentum compared to L. pennellii, whereas the opposite response was obtained in calluses. The results obtained in this study suggest that rooting parameters are the most useful traits for rapid evaluation and screening of tomato species and segregating populations through in vitro shoot apex culture. This revised version was published online in June 2006 with corrections to the Cover Date.     

Song sparrows, top carnivores and nest predation: a test of the mesopredator release hypothesis

Ground-nesting North American landbirds have declined in the longterm, including species with a variety of migratory strategies. The mesopredator release hypothesis explains declines by suggesting that the virtual elimination of top carnivores (large-bodied canids and felids) from much of North America has “released” populations of nest-destroying mesopredators (i.e., medium-sized terrestrial omnivores such as the raccoon Procyon lotor). The hypothesis predicts (1) higher nest success in the presence than in the absence of top carnivores, and (2) a positive relationship between mesopredator abundance and nest predation. Results from a 4-year "natural experiment" in the agricultural landscape of southern Michigan tended to support prediction 1. When coyotes (Canis latrans) were absent in 1993 and present in 1994, 1995 and 1996, Mayfield nest survival in song sparrows (Melospiza melodia), a ground-nesting landbird, increased significantly during the same 4-year interval. Relative frequency of nest predation, the most common cause of nest failure, declined significantly over the four years. Coyotes may have decreased nest predation in 1994–1996 by reducing the abundance of raccoons, apparently the main nest predator in the study area. In an experiment testing prediction 2, mesopredator abundance and predation rate on artificial nests were positively related, as predicted by the hypothesis. Although the present study is not wholly conclusive by itself, we cautiously suggest it contributes to a growing body of evidence derived from a number of studies supporting the mesopredator release hypothesis. Received: 23 May 1997 / Accepted: 12 January 1998