A simple method to introduce marker-free genetic modifications into the chromosome of naturally nontransformable Bacillus amyloliquefaciens strains

A simple method to introduce marker-free deletions, insertions, and point mutations into the chromosomes of naturally nontransformable Bacillus amyloliquefaciens strains has been developed. The method is efficient and fast, and it allows for the generation of genetic modifications without the use of a counter-selectable marker or a special prerequisite strain. This method uses the combination of the following: the effective introduction of a delivery plasmid into cells for gene replacement; a two-step replacement recombination procedure, which occurs at a very high frequency due to the use of a thermosensitive rolling-circle replication plasmid; and colony polymerase chain reaction (PCR) analysis for screening. Using PCR primers with mismatches at the 3′ end enables the selection of strains that contain a single nucleotide substitution in the target gene. This approach can be used as a routine method for the investigation of complex physiological pathways and for the metabolic engineering of food-grade industrial B. amyloliquefaciens and other Bacillus strains.     

Temperature-regulated formation of mycelial mat-like biofilms by Legionella pneumophila

Fifty strains representing 38 species of the genus Legionella were examined for biofilm formation on glass, polystyrene, and polypropylene surfaces in static cultures at 25 degrees C, 37 degrees C, and 42 degrees C. Strains of Legionella pneumophila, the most common causative agent of Legionnaires' disease, were found to have the highest ability to form biofilms among the test strains. The quantity, rate of formation, and adherence stability of L. pneumophila biofilms showed considerable dependence on both temperature and surface material. Glass and polystyrene surfaces gave between two- to sevenfold-higher yields of biofilms at 37 degrees C or 42 degrees C than at 25 degrees C; conversely, polypropylene surface had between 2 to 16 times higher yields at 25 degrees C than at 37 degrees C or 42 degrees C. On glass surfaces, the biofilms were formed faster but attached less stably at 37 degrees C or 42 degrees C than at 25 degrees C. Both scanning electron microscopy and confocal laser scanning microscopy revealed that biofilms formed at 37 degrees C or 42 degrees C were mycelial mat like and were composed of filamentous cells, while at 25 degrees C, cells were rod shaped. Planktonic cells outside of biofilms or in shaken liquid cultures were rod shaped. Notably, the filamentous cells were found to be multinucleate and lacking septa, but a recA null mutant of L. pneumophila was unaffected in its temperature-regulated filamentation within biofilms. Our data also showed that filamentous cells were able to rapidly give rise to a large number of short rods in a fresh liquid culture at 37 degrees C. The possibility of this biofilm to represent a novel strategy by L. pneumophila to compete for proliferation among the environmental microbiota is discussed.     

Short-term fasting, seizure control and brain amino acid metabolism

The ketogenic diet is an effective treatment for seizures, but the mechanism of action is unknown. It is uncertain whether the anti-epileptic effect presupposes ketosis, or whether the restriction of calories and/or carbohydrate might be sufficient. We found that a relatively brief (24 h) period of low glucose and low calorie intake significantly attenuated the severity of seizures in young Sprague-Dawley rats (50-70 gms) in whom convulsions were induced by administration of pentylenetetrazole (PTZ). The blood glucose concentration was lower in animals that received less dietary glucose, but the brain glucose level did not differ from control blood [3-OH-butyrate] tended to be higher in blood, but not in brain, of animals on a low-glucose intake. The concentration in brain of glutamine increased and that of alanine declined significantly with low-glucose intake. The blood alanine level fell more than that of brain alanine, resulting in a marked increase ( approximately 50%) in the brain:blood ratio for alanine. In contrast, the brain:blood ratio for leucine declined by about 35% in the low-glucose group. When animals received [1-(13)C]glucose, a metabolic precursor of alanine, the appearance of (13)C in alanine and glutamine increased significantly relative to control. The brain:blood ratio for [(13)C]alanine exceeded 1, indicating that the alanine must have been formed in brain and not transported from blood. The elevated brain(alanine):blood(alanine) could mean that a component of the anti-epileptic effect of low carbohydrate intake is release of alanine from brain-to-blood, in the process abetting the disposal of glutamate, excess levels of which in the synaptic cleft would contribute to the development of seizures.     

Detection of presenilin-1 homodimer formation in intact cells using fluorescent lifetime imaging microscopy

Presenilin-1 (PS1) is a multipass transmembrane domain protein, which is believed to be the catalytic component of the gamma-secretase complex. The complex is comprised of four major components: PS1, nicastrin, Aph-1, and Pen-2. The exact stoichiometric relationship between the four components remains unclear. It has been shown that gamma-secretase exists as high molecular weight complexes, suggesting the possibility of dimer/multimer formation. We combined a biochemical approach with a novel morphological microscopy assay to analyze PS1 dimer formation and subcellular distribution in situ, in intact mammalian cells. Both coimmunoprecipitation and fluorescent lifetime imaging microscopy approaches showed that wildtype PS1 molecules form dimers. Moreover, PS1 holoproteins containing the D257A mutation also come into close enough proximity to form a dimer, suggesting that cleavage within the loop is not necessary for dimer formation. Taken together these data suggest that PS1 dimerization occurs during normal PS1 function.     

Temperature-Regulated Formation of Mycelial Mat-Like Biofilms by Legionella pneumophila

Fifty strains representing 38 species of the genus Legionella were examined for biofilm formation on glass, polystyrene, and polypropylene surfaces in static cultures at 25°C, 37°C, and 42°C. Strains of Legionella pneumophila, the most common causative agent of Legionnaires' disease, were found to have the highest ability to form biofilms among the test strains. The quantity, rate of formation, and adherence stability of L. pneumophila biofilms showed considerable dependence on both temperature and surface material. Glass and polystyrene surfaces gave between two- to sevenfold-higher yields of biofilms at 37°C or 42°C than at 25°C; conversely, polypropylene surface had between 2 to 16 times higher yields at 25°C than at 37°C or 42°C. On glass surfaces, the biofilms were formed faster but attached less stably at 37°C or 42°C than at 25°C. Both scanning electron microscopy and confocal laser scanning microscopy revealed that biofilms formed at 37°C or 42°C were mycelial mat like and were composed of filamentous cells, while at 25°C, cells were rod shaped. Planktonic cells outside of biofilms or in shaken liquid cultures were rod shaped. Notably, the filamentous cells were found to be multinucleate and lacking septa, but a recA null mutant of L. pneumophila was unaffected in its temperature-regulated filamentation within biofilms. Our data also showed that filamentous cells were able to rapidly give rise to a large number of short rods in a fresh liquid culture at 37°C. The possibility of this biofilm to represent a novel strategy by L. pneumophila to compete for proliferation among the environmental microbiota is discussed.     

The alternative stimulatory G protein alpha-subunit XLalphas is a critical regulator of energy and glucose metabolism and sympathetic nerve activity in adult mice

The complex imprinted Gnas locus encodes several gene products including G(s)alpha, the ubiquitously expressed G protein alpha-subunit required for receptor-stimulated cAMP generation, and the neuroendocrine-specific G(s)alpha isoform XLalphas. XLalphas is only expressed from the paternal allele, whereas G(s)alpha is biallelically expressed in most tissues. XLalphas knock-out mice (Gnasxl(m+/p-)) have poor suckling and perinatal lethality, implicating XLalphas as critical for postnatal feeding. We have now examined the metabolic phenotype of adult Gnasxl(m+/p-) mice. Gnasxl(m+/p-) mice had reduced fat mass and lipid accumulation in adipose tissue, with increased food intake and metabolic rates. Gene expression profiling was consistent with increased lipid metabolism in adipose tissue. These changes likely result from increased sympathetic nervous system activity rather than adipose cell-autonomous effects, as we found that XLalphas is not normally expressed in adult adipose tissue, and Gnasxl(m+/p-) mice had increased urinary norepinephrine levels but not increased metabolic responsiveness to a beta3-adrenergic agonist. Gnasxl(m+/p-) mice were hypolipidemic and had increased glucose tolerance and insulin sensitivity. The similar metabolic profile observed in some prior paternal Gnas knock-out models results from XLalphas deficiency (or deficiency of the related alternative truncated protein XLN1). XLalphas (or XLN1) is a negative regulator of sympathetic nervous system activity in mice.     

Qualitative and quantitative characteristics of the extracellular DNA delivered to the nucleus of a living cell

Background  

The blood plasma and other intertissue fluids usually contain a certain amount of DNA, getting there due to a natural cell death in the organism. Cells of this organism can capture the extracellular DNA, whereupon it is delivered to various cell compartments. It is hypothesized that the extracellular DNA is involved in the transfer of genetic information and its fixation in the genome of recipient cell.     

Persistent cytomegalovirus-specific memory responses in the lung allograft and blood following primary infection in lung transplant recipients

Primary CMV infection in lung transplant recipients (LTRs) is associated with increased mortality. We studied 22 donor CMV-positive, recipient-negative (D(+)R(-)) LTRs for the development of posttransplant CMV-specific immunity. We found that 13 of 22 D(+)R(-) LTRs (59.1%) seroconverted (CMV IgG Ab(+)). Using pooled peptides of the immunodominant CMV Ags pp65 and IE1, we detected CMV-specific CD8(+)IFN-gamma(+) T cells in the PBMC of 90% of seroconverted individuals following primary infection by intracellular cytokine staining. In contrast, few seroconverters had detectable CMV-specific CD4(+)IFN-gamma(+) T cells during viral latency. However, the majority of IgG(+) LTRs demonstrated CMV-specific CD4(+) and CD8(+) T cell proliferative responses from PBMC, with CD4(+)IFN-gamma(+) T cells detectable upon re-expansion. Examination of lung allograft mononuclear cells obtained by bronchoalveolar lavage revealed both CMV-specific CD4(+) and CD8(+)IFN-gamma(+) T cells, including patients from whom CD4(+)IFN-gamma(+) T cells were simultaneously undetectable in the PBMC, suggesting differential effector memory populations between these compartments. Moreover, both responses in the PBMC and lung allograft were found to persist, despite substantial immunosuppression, long after primary infection. Clinical correlation in this cohort demonstrated that the acquisition of CMV immunity was associated with freedom from CMV disease (p < or = 0.009) and preservation of allograft function (p < or = 0.02) compared with those who failed to develop CMV immunity. Together, our data reveal immunologic heterogeneity in D(+)R(-) LTRs, with the development and persistence of primary CMV responses that may provide clinical benefit.     

New Insight into Erythrocyte through In Vivo Surface-Enhanced Raman Spectroscopy

The article presents a noninvasive approach to the study of erythrocyte properties by means of a comparative analysis of signals obtained by surface-enhanced Raman spectroscopy (SERS) and resonance Raman spectroscopy (RS). We report step-by-step the procedure for preparing experimental samples containing erythrocytes in their normal physiological environment in a mixture of colloid solution with silver nanoparticles and the procedure for the optimization of SERS conditions to achieve high signal enhancement without affecting the properties of living erythrocytes. By means of three independent techniques, we demonstrate that under the proposed conditions a colloid solution of silver nanoparticles does not affect the properties of erythrocytes. For the first time to our knowledge, we describe how to use the SERS-RS approach to study two populations of hemoglobin molecules inside an intact living erythrocyte: submembrane and cytosolic hemoglobin (Hb_sm and Hb_c). We show that the conformation of Hb_sm differs from the conformation of Hb_c. This finding has an important application, as the comparative study of Hb_sm and Hb_c could be successfully used in biomedical research and diagnostic tests.     

Mild cholesterol depletion reduces amyloid-β production by impairing APP trafficking to the cell surface

It has been suggested that cellular cholesterol levels can modulate the metabolism of the amyloid precursor protein (APP) but the underlying mechanism remains controversial. In the current study, we investigate in detail the relationship between cholesterol reduction, APP processing and γ-secretase function in cell culture studies. We found that mild membrane cholesterol reduction led to a decrease in Aβ₄₀ and Aβ₄₂ in different cell types. We did not detect changes in APP intracellular domain or Notch intracellular domain generation. Western blot analyses showed a cholesterol-dependent decrease in the APP C-terminal fragments and cell surface APP. Finally, we applied a fluorescence resonance energy transfer (FRET)-based technique to study APP–Presenilin 1 (PS1) interactions and lipid rafts in intact cells. Our data indicate that cholesterol depletion reduces association of APP into lipid rafts and disrupts APP–PS1 interaction. Taken together, our results suggest that mild membrane cholesterol reduction impacts the cleavage of APP upstream of γ-secretase and appears to be mediated by changes in APP trafficking and partitioning into lipid rafts.