Seasonal influence on heat-resistant proteolytic capacity of Pseudomonas lundensis and Pseudomonas fragi, predominant milk spoilers isolated from Belgian raw milk samples

Psychrotolerant bacteria and their heat-resistant proteases play a major role in the spoilage of UHT-processed dairy products. Summer and winter raw milk samples were screened for the presence of such bacteria. One hundred and three proteolytic psychrotolerant bacteria were isolated, characterized by API tests, rep-PCR fingerprint analysis and evaluated for heat-resistant protease production. Twenty-nine strains (representing 79% of the complete collection) were further identified by 16S rRNA gene sequencing, rpoB gene sequencing and DNA–DNA hybridizations. A seasonal inter- and intra-species influence on milk spoilage capacity (e.g. growth rate and/or protease production) was demonstrated. Moreover, this polyphasic approach led to the identification of Pseudomonas fragi and Pseudomonas lundensis (representing 53% of all isolates) as predominant producers of heat-resistant proteases in raw milk. The role of Pseudomonas fluorescens , historically reported as important milk spoiler, could not unequivocally be established. The use of more reliable identification techniques and further revision of the taxonomy of P. fluorescens will probably result in a different perspective on its role in the milk spoilage issue.     

Increased Lipid Accumulation in the Chlamydomonas reinhardtii sta7-10 Starchless Isoamylase Mutant and Increased Carbohydrate Synthesis in Complemented Strains

The accumulation of bioenergy carriers was assessed in two starchless mutants of Chlamydomonas reinhardtii (the sta6 [ADP-glucose pyrophosphorylase] and sta7-10 [isoamylase] mutants), a control strain (CC124), and two complemented strains of the sta7-10 mutant. The results indicate that the genetic blockage of starch synthesis in the sta6 and sta7-10 mutants increases the accumulation of lipids on a cellular basis during nitrogen deprivation relative to that in the CC124 control as determined by conversion to fatty acid methyl esters. However, this increased level of lipid accumulation is energetically insufficient to completely offset the loss of cellular starch that is synthesized by CC124 during nitrogen deprivation. We therefore investigated acetate utilization and O₂ evolution to obtain further insights into the physiological adjustments utilized by the two starchless mutants in the absence of starch synthesis. The results demonstrate that both starchless mutants metabolize less acetate and have more severely attenuated levels of photosynthetic O₂ evolution than CC124, indicating that a decrease in overall anabolic processes is a significant physiological response in the starchless mutants during nitrogen deprivation. Interestingly, two independent sta7-10:STA7 complemented strains exhibited significantly greater quantities of cellular starch and lipid than CC124 during acclimation to nitrogen deprivation. Moreover, the complemented strains synthesized significant quantities of starch even when cultured in nutrient-replete medium.Microalgae are able to efficiently convert sunlight, water, and CO₂ into a variety of products suitable for renewable energy applications, including H₂, carbohydrates, and lipids (11, 12, 16, 21, 38, 41, 44). The unicellular green alga Chlamydomonas reinhardtii has emerged as a model organism for studying algal physiology, photosynthesis, metabolism, nutrient stress, and the synthesis of bioenergy carriers (12, 15, 19, 24, 32). During acclimation to nitrogen deprivation, C. reinhardtii cells accumulate significant quantities of starch and form lipid bodies (4, 5, 8, 26, 28, 30, 34, 43, 46, 48). Despite the significance of these products in algal physiology and in biofuels applications, the metabolic, enzymatic, and regulatory mechanisms controlling the partitioning of metabolites into these distinct carbon stores in algae are poorly understood. Several C. reinhardtii starch mutants with various phenotypic changes in starch content and structure have been isolated (2,–4). Two of these, the sta6 and sta7 mutants, contain single-gene disruptions that result in “starchless” phenotypes with severely attenuated levels of starch granule accumulation (2, 4, 34, 39, 40, 48).The disrupted loci in the two isolated starchless mutants are distinct and each mutant has a unique phenotype (7, 40). In the sta6 mutant, the small, catalytic subunit of ADP-glucose pyrophosphorylase (AGPase-SS) is disrupted (2, 4, 48), and this mutant accumulates less than 1% of the starch observed in wild-type (WT) cells under conditions of nitrogen deprivation. The sta7 mutant contains a disrupted isoamylase gene (7, 8, 10, 39, 40) and also has severely attenuated levels of starch, but it accumulates a soluble glycogen-like product (4, 9). In this study, we conducted an examination of the unique physiological acclimations that are utilized by these mutants to adapt to the loss of starch synthesis. As the genetic lesions in these two mutants are distinct and block starch synthesis via two very different mechanisms, we investigated the physiological consequences of starch inhibition in both of these mutants from a holistic bioenergy perspective, which included photosynthetic parameters and the overall yields of lipids and carbohydrates, the two primary bioenergy carriers in C. reinhardtii. Specifically, we examined whether the inability to synthesize starch would result in the accumulation of additional lipid, alter cellular growth or cell size, affect acetate utilization, and/or influence photosynthetic O₂ evolution. Our data indicate that both the sta6 (BAFJ5) and sta7 (sta7-10) mutants accumulate more lipid than the CC124 control during nitrogen deprivation. However, the additional lipid does not completely offset the loss of starch synthesis from a complete energetic perspective. Increased lipid accumulation during nitrogen stress has also been reported for a variety of starch mutants in recent papers (26, 27, 46). A significant feature in both of the starchless mutants studied here is that O₂ evolution and acetate utilization are diminished during nitrogen stress, which is undesirable from an overall bioenergy perspective. Remarkably, complementation of sta7-10 with genomic DNA encoding the wild-type isoamylase gene resulted in cells that were larger than those of the sta6, sta7-10, and CC124 strains, exhibited the highest total lipid levels during nitrogen deprivation, and overaccumulated starch even in nutrient-replete medium.     

Human herpesvirus 6 infection arrests cord blood mononuclear cells in G(2) phase of the cell cycle

We here report that after infection with human herpesvirus 6A, human cord blood mononuclear cells accumulate in G₂/M phase of the cell cycle. Experiments with foscarnet or ultraviolet (UV)-irradiated virus stocks pointed at an (immediate-)early, newly formed viral protein to be responsible for the arrest. At the molecular level, p53, cyclin B₁, cyclin A and tyrosine¹⁵-phosphorylated cdk1 accumulated after HHV-6A infection, indicating an arrest in G₂. However, no change was observed in the levels of downstream effectors of p53 in establishing a G₂ arrest, i.e. p21 and 14-3-3σ. We thus conclude that the HHV-6A-induced G₂ arrest occurs independently of p53 accumulation.     

A putative twin-arginine translocation pathway in Legionella pneumophila

Legionella pneumophila is a facultative intracellular human pathogen causing Legionnaires' disease, a severe form of pneumonia. Because of the importance of secretion pathways in virulence, we were interested in the possible presence of the twin-arginine translocation (Tat) pathway in L. pneumophila. This secretion pathway is used to transport folded proteins, characterized by two arginines in their signal peptide, across the cytoplasmic membrane. We describe here the presence of a putative Tat pathway in L. pneumophila. Three genes encoding Escherichia coli TatA, TatB, and TatC homologues were identified. The tatA and tatB genes were shown to constitute an operon while tatC is monocistronic. RT-PCR analysis revealed expression of the tat genes during both exponential and stationary growth as well as during intracellular growth in Acanthamoeba castellanii. A search for the conserved twin-arginine motif in predicted signal peptides resulted in a list of putative Tat substrates.     

Characterization of amyloid beta peptides from brain extracts of transgenic mice overexpressing the London mutant of human amyloid precursor protein

Alzheimer's disease (AD) is marked by the presence of neurofibrillary tangles and amyloid plaques in the brain of patients. To study plaque formation, we report on further quantitative and qualitative analysis of human and mouse amyloid beta peptides (Abeta) from brain extracts of transgenic mice overexpressing the London mutant of human amyloid precursor protein (APP). Using enzyme-linked immunosorbant assays (ELISAs) specific for either human or rodent Abeta, we found that the peptides from both species aggregated to form plaques. The ratios of deposited Abeta1-42/1-40 were in the order of 2-3 for human and 8-9 for mouse peptides, indicating preferential deposition of Abeta42. We also determined the identity and relative levels of other Abeta variants present in protein extracts from soluble and insoluble brain fractions. This was done by combined immunoprecipitation and mass spectrometry (IP/MS). The most prominent peptides truncated either at the carboxyl- or the amino-terminus were Abeta1-38 and Abeta11-42, respectively, and the latter was strongly enriched in the extracts of deposited peptides. Taken together, our data indicate that plaques of APP-London transgenic mice consist of aggregates of multiple human and mouse Abeta variants, and the human variants that we identified were previously detected in brain extracts of AD patients.     

Analogs of 1alpha,25-dihydroxyvitamin D3 as pluripotent immunomodulators

The active form of vitamin D(3), 1,25(OH)(2)D(3), is known, besides its classical effects on calcium and bone, for its pronounced immunomodulatory effects that are exerted both on the antigen-presenting cell level as well as directly on the T lymphocyte level. In animal models, these immune effects of 1,25(OH)(2)D(3) are reflected by a strong potency to prevent onset and even recurrence of autoimmune diseases. A major limitation in using 1,25(OH)(2)D(3) in clinical immune therapy are the adverse side effects on calcium and on bone. TX527 (19-nor-14,20-bisepi-23-yne-1,25(OH)(2)D(3)) is a structural 1,25(OH)(2)D(3) analog showing reduced calcemic activity associated with enhanced in vitro and in vivo immunomodulating capacity compared to the mother-molecule. Indeed, in vitro TX527 is more potent that 1,25(OH)(2)D(3) in redirecting differentiation and maturation of dendritic cells and in inhibiting phytohemagglutinin-stimulated T lymphocyte proliferation. In vivo, this enhanced potency of TX527 is confirmed by a stronger potential to prevent type 1 diabetes in nonobese diabetic (NOD) mice and to prolong the survival of syngeneic islets grafts, both alone and in combination with cyclosporine A, in overtly diabetic NOD mice. Moreover, these in vivo effects of TX527 are obtained without the adverse side effects observed for 1,25(OH)(2)D(3) itself. We believe therefore that TX527 is a potentially interesting candidate to be considered for clinical intervention trails in autoimmune diseases.     

VEGF: a modifier of the del22q11 (DiGeorge) syndrome?

Hemizygous deletion of chromosome 22q11 (del22q11) causes thymic, parathyroid, craniofacial and life-threatening cardiovascular birth defects in 1 in 4,000 infants. The del22q11 syndrome is likely caused by haploinsufficiency of TBX1, but its variable expressivity indicates the involvement of additional modifiers. Here, we report that absence of the Vegf164 isoform caused birth defects in mice, reminiscent of those found in del22q11 patients. The close correlation of birth and vascular defects indicated that vascular dysgenesis may pathogenetically contribute to the birth defects. Vegf interacted with Tbx1, as Tbx1 expression was reduced in Vegf164-deficient embryos and knocked-down vegf levels enhanced the pharyngeal arch artery defects induced by tbx1 knockdown in zebrafish. Moreover, initial evidence suggested that a VEGF promoter haplotype was associated with an increased risk for cardiovascular birth defects in del22q11 individuals. These genetic data in mouse, fish and human indicate that VEGF is a modifier of cardiovascular birth defects in the del22q11 syndrome.     

Revascularization of ischemic tissues by PlGF treatment,and inhibition of tumor angiogenesis,arthritis and atherosclerosis by anti-Flt1

The therapeutic potential of placental growth factor (PlGF) and its receptor Flt1 in angiogenesis is poorly understood. Here, we report that PlGF stimulated angiogenesis and collateral growth in ischemic heart and limb with at least a comparable efficiency to vascular endothelial growth factor (VEGF). An antibody against Flt1 suppressed neovascularization in tumors and ischemic retina, and angiogenesis and inflammatory joint destruction in autoimmune arthritis. Anti-Flt1 also reduced atherosclerotic plaque growth and vulnerability, but the atheroprotective effect was not attributable to reduced plaque neovascularization. Inhibition of VEGF receptor Flk1 did not affect arthritis or atherosclerosis, indicating that inhibition of Flk1-driven angiogenesis alone was not sufficient to halt disease progression. The anti-inflammatory effects of anti-Flt1 were attributable to reduced mobilization of bone marrow-derived myeloid progenitors into the peripheral blood; impaired infiltration of Flt1-expressing leukocytes in inflamed tissues; and defective activation of myeloid cells. Thus, PlGF and Flt1 constitute potential candidates for therapeutic modulation of angiogenesis and inflammation.