Mutations in the NB-ARC domain of I-2 that impair ATP hydrolysis cause autoactivation

Resistance (R) proteins in plants confer specificity to the innate immune system. Most R proteins have a centrally located NB-ARC (nucleotide-binding adaptor shared by APAF-1, R proteins, and CED-4) domain. For two tomato (Lycopersicon esculentum) R proteins, I-2 and Mi-1, we have previously shown that this domain acts as an ATPase module that can hydrolyze ATP in vitro. To investigate the role of nucleotide binding and hydrolysis for the function of I-2 in planta, specific mutations were introduced in conserved motifs of the NB-ARC domain. Two mutations resulted in autoactivating proteins that induce a pathogen-independent hypersensitive response upon expression in planta. These mutant forms of I-2 were found to be impaired in ATP hydrolysis, but not in ATP binding, suggesting that the ATP- rather than the ADP-bound state of I-2 is the active form that triggers defense signaling. In addition, upon ADP binding, the protein displayed an increased affinity for ADP suggestive of a change of conformation. Based on these data, we propose that the NB-ARC domain of I-2, and likely of related R proteins, functions as a molecular switch whose state (on/off) depends on the nucleotide bound (ATP/ADP).     

Inhibitory antibodies to human angiotensin-converting enzyme: fine epitope mapping and mechanism of action

Angiotensin I-converting enzyme (ACE), a key enzyme in cardiovascular pathophysiology, consists of two homologous domains (N and C), each bearing a Zn-dependent active site. We modeled the 3D-structure of the ACE N-domain using known structures of the C-domain of human ACE and the ACE homologue, ACE2, as templates. Two monoclonal antibodies (mAb), 3A5 and i2H5, developed against the human N-domain of ACE, demonstrated anticatalytic activity. N-domain modeling and mutagenesis of 21 amino acid residues allowed us to define the epitopes for these mAbs. Their epitopes partially overlap: amino acid residues K407, E403, Y521, E522, G523, P524, D529 are present in both epitopes. Mutation of 4 amino acid residues within the 3A5 epitope, N203E, R550A, D558L, and K557Q, increased the apparent binding of mAb 3A5 with the mutated N-domain 3-fold in plate precipitation assay, but abolished the inhibitory potency of this mAb. Moreover, mutation D558L dramatically decreased 3A5-induced ACE shedding from the surface of CHO cells expressing human somatic ACE. The inhibition of N-domain activity by mAbs 3A5 and i2H5 obeys similar kinetics. Both mAbs can bind to the free enzyme and enzyme-substrate complex, forming E.mAb and E.S.mAb complexes, respectively; however, only complex E.S can form a product. Kinetic analysis indicates that both mAbs bind better with the ACE N-domain in the presence of a substrate, which, in turn, implies that binding of a substrate causes conformational adjustments in the N-domain structure. Independent experiments with ELISA demonstrated better binding of mAbs 3A5 and i2H5 in the presence of the inhibitor lisinopril as well. This effect can be attributed to better binding of both mAbs with the "closed" conformation of ACE, therefore, disturbing the hinge-bending movement of the enzyme, which is necessary for catalysis.     

Repeated intraspecific divergence in life span and aging of African annual fishes along an aridity gradient

Life span and aging are substantially modified by natural selection. Across species, higher extrinsic (environmentally related) mortality (and hence shorter life expectancy) selects for the evolution of more rapid aging. However, among populations within species, high extrinsic mortality can lead to extended life span and slower aging as a consequence of condition‐dependent survival. Using within‐species contrasts of eight natural populations of Nothobranchius fishes in common garden experiments, we demonstrate that populations originating from dry regions (with short life expectancy) had shorter intrinsic life spans and a greater increase in mortality with age, more pronounced cellular and physiological deterioration (oxidative damage, tumor load), and a faster decline in fertility than populations from wetter regions. This parallel intraspecific divergence in life span and aging was not associated with divergence in early life history (rapid growth, maturation) or pace‐of‐life syndrome (high metabolic rates, active behavior). Variability across four study species suggests that a combination of different aging and life‐history traits conformed with or contradicted the predictions for each species. These findings demonstrate that variation in life span and functional decline among natural populations are linked, genetically underpinned, and can evolve relatively rapidly.     

Identification of human-pathogenic strains of Shiga toxin-producing Escherichia coli from food by a combination of serotyping and molecular typing of Shiga toxin genes

We examined 219 Shiga toxin-producing Escherichia coli (STEC) strains from meat, milk, and cheese samples collected in Germany between 2005 and 2006. All strains were investigated for their serotypes and for genetic variants of Shiga toxins 1 and 2 (Stx1 and Stx2). stx(1) or variant genes were detected in 88 (40.2%) strains and stx(2) and variants in 177 (80.8%) strains. Typing of stx genes was performed by stx-specific PCRs and by analysis of restriction fragment length polymorphisms (RFLP) of PCR products. Major genotypes of the Stx1 (stx(1), stx(1c), and stx(1d)) and the Stx2 (stx(2), stx(2d), stx(2-O118), stx(2e), and stx(2g)) families were detected, and multiple types of stx genes coexisted frequently in STEC strains. Only 1.8% of the STEC strains from food belonged to the classical enterohemorrhagic E. coli (EHEC) types O26:H11, O103:H2, and O157:H7, and only 5.0% of the STEC strains from food were positive for the eae gene, which is a virulence trait of classical EHEC. In contrast, 95 (43.4%) of the food-borne STEC strains carried stx(2) and/or mucus-activatable stx(2d) genes, an indicator for potential high virulence of STEC for humans. Most of these strains belonged to serotypes associated with severe illness in humans, such as O22:H8, O91:H21, O113:H21, O174:H2, and O174:H21. stx(2) and stx(2d) STEC strains were found frequently in milk and beef products. Other stx types were associated more frequently with pork (stx(2e)), lamb, and wildlife meat (stx(1c)). The combination of serotyping and stx genotyping was found useful for identification and for assignment of food-borne STEC to groups with potential lower and higher levels of virulence for humans.     

Importance of changes in gastric emptying for postprandial plasma glucose fluxes in healthy humans

Angiotensin II (Ang II) stimulation of the Ang type 1 receptor (AT(1)R) facilitates myocardial remodeling through NADPH oxidase-mediated generation of oxidative stress. Components of the renin-angiotensin system constitute an autocrine/paracrine unit in the myocardium, including renin, which is the rate-limiting step in the generation of Ang II. This investigation sought to determine whether cardiac oxidative stress and cellular remodeling could be attenuated by in vivo renin inhibition and/or AT(1)R blockade in a rodent model of chronically elevated tissue Ang II levels, the transgenic (mRen2)27 rat (Ren2). The Ren2 overexpresses the mouse renin transgene with resultant hypertension, insulin resistance, and cardiovascular damage. Young (6- to 7-wk-old) heterozygous (+/-) male Ren2 and age-matched Sprague-Dawley rats were treated with the renin inhibitor aliskiren, which has high preferential affinity for human and mouse renin, an AT(1)R blocker, irbesartan, or placebo for 3 wk. Myocardial NADPH oxidase activity and immunostaining for NADPH oxidase subunits and 3-nitrotyrosine were evaluated and remodeling changes assessed by light and transmission electron microscopy. Blood pressure, myocardial NADPH oxidase activity and subunit immunostaining, 3-nitrotyrosine, perivascular fibrosis, mitochondrial content, and markers of activity were significantly increased in Ren2 compared with SD littermates. Both renin inhibition and blockade of the AT(1)R significantly attenuated cardiac functional and structural alterations, although irbesartan treatment resulted in greater reductions of both blood pressure and markers of oxidative stress. Collectively, these data suggest that both reduce changes driven, in part, by Ang II-mediated increases in NADPH oxidase and, in part, increases in blood pressure.     

Impact of the FcgammaII-receptor on quartz uptake and inflammatory response by alveolar macrophages

The inflammatory response following particle inhalation is described as a key event in the development of lung diseases, e.g., fibrosis and cancer. The essential role of alveolar macrophages (AM) in the pathogenicity of particles through their functions in lung clearance and mediation of inflammation is well known. However, the molecular mechanisms and direct consequences of particle uptake are still unclear. Inhibition of different classic phagocytosis receptors by flow cytometry shows a reduction of the dose-dependent quartz particle (DQ12) uptake in the rat AM cell line NR8383. Thereby the strongest inhibitory effect was observed by blocking the FcgammaII-receptor (FcgammaII-R). Fluorescence immunocytochemistry, demonstrating FcgammaII-R clustering at particle binding sites as well as transmission electron microscopy, visualizing zippering mechanism-like morphological changes, confirmed the role of the FcgammaII-R in DQ12 phagocytosis. FcgammaII-R participation in DQ12 uptake was further strengthened by the quartz-induced activation of the Src-kinase Lyn, the phospho-tyrosine kinases Syk (spleen tyrosine kinase) and PI3K (phosphatidylinositol 3-kinase), as shown by Western blotting. Activation of the small GTPases Rac1 and Cdc42, shown by immunoprecipitation, as well as inhibition of tyrosine kinases, GTPases, or Rac1 provided further support for the role of the FcgammaII-R. Consistent with the uptake results, FcgammaII-R activation with its specific ligand caused a similar generation of reactive oxygen species and TNF-alpha release as observed after treatment with DQ12. In conclusion, our results indicate a major role of FcgammaII-R and its downstream signaling cascade in the phagocytosis of quartz particles in AM as well as in the associated generation and release of inflammatory mediators.     

Influence of ELF sinusoidal electromagnetic fields on proliferation and metabolite yield of fungi

The response of mycelium proliferation in 12 strains of fungi were tested by sinusoidal ELF 50 Hz electromagnetic field treatment in the range B = 0.6-10 mT over 10 days. The ratio of experiment/control indicated three types of proliferation changes: a) no significant change, b) a strong decrease down to E/C = 0.2, c) a maximization of mycelium diameter by treatment at 5-7 mT. According to these results, effects can be produced noninvasively by varying either magnetic intensities or time of treatment. As yet, systematic bioelectromagnetic research using sinusoidal electromagnetic fields (SEMF)-on fermentation of fungi is still in its initial stages.     

Determinants of Brushite Stone Formation: A Case-Control Study

Purpose

The occurrence of brushite stones has increased during recent years. However, the pathogenic factors driving the development of brushite stones remain unclear.

Methods

Twenty-eight brushite stone formers and 28 age-, sex- and BMI-matched healthy individuals were enrolled in this case-control study. Anthropometric, clinical, 24 h urinary parameters and dietary intake from 7-day weighed food records were assessed.

Results

Pure brushite stones were present in 46% of patients, while calcium oxalate was the major secondary stone component. Urinary pH and oxalate excretion were significantly higher, whereas urinary citrate was lower in patients as compared to healthy controls. Despite lower dietary intake, urinary calcium excretion was significantly higher in brushite stone patients. Binary logistic regression analysis revealed pH>6.50 (OR 7.296; p = 0.035), calcium>6.40 mmol/24 h (OR 25.213; p = 0.001) and citrate excretion <2.600 mmol/24 h (OR 15.352; p = 0.005) as urinary risk factors for brushite stone formation. A total of 56% of patients exhibited distal renal tubular acidosis (dRTA). Urinary pH, calcium and citrate excretion did not significantly differ between patients with or without dRTA.

Conclusions

Hypercalciuria, a diminished citrate excretion and an elevated pH turned out to be the major urinary determinants of brushite stone formation. Interestingly, urinary phosphate was not associated with urolithiasis. The increased urinary oxalate excretion, possibly due to decreased calcium intake, promotes the risk of mixed stone formation with calcium oxalate. Neither dietary factors nor dRTA can account as cause for hypercalciuria, higher urinary pH and diminished citrate excretion. Further research is needed to define the role of dRTA in brushite stone formation and to evaluate the hypothesis of an acquired acidification defect.     

Clonal domains in postlarval Platynereis dumerilii (Annelida: Polychaeta)

Following an enzymatic procedure for softening the egg envelope, blastomeres in the embryo of the polychaete Platynereis dumerilii were injected with TRITC-dextran. Injection was successful in the following blastomeres: AB, CD, A, B, C, D, 1a-1d, 1A-1D, 4d, and 4d(1). The distribution of fluorescent label was recorded by confocal laser scanning microscopy of young, three-segmented worms after 3 or 4 days of development, in some cases also in 1-day-old trochophore larvae. Results were documented by single optical sections, by stacking a limited number or a complete set of optical sections, and by computer-generated surface views of both the labeled tissue domains and the body contours from complete image stacks of whole worms. With respect to their descent from the embryonic cell pattern, five major compartments can be distinguished which together compose the body of the young worm: 1) The epispheric, epidermal, and neural region of the head, composed of four domains arranged as quasi-radial sectors derived from micromeres 1a, 1b (left and right ventral), and 1c and 1d (right and left dorsal). 2) A posttrochal epidermal region of the head originating from micromeres 2a(1)-2c(1) and constituting the ventral and lateral posttrochal epidermis of the head. 3) A stomodeal-ectomesodermal region of the head, including the stomodeum (micromeres 2a(2) and 2c(2)), its mesodermal envelope, and head mesoderm (micromeres 3a-3d). 4) A solid cone composed of the four terminal macromeres 4A-4D, forming the core of the trunk as the endoderm anlage. 5) An epidermal and mesodermal coating of the trunk originating from the dorsal micromeres 2d and 4d. The region of the so-called (first, anterior) peristomial cirri at the posterior flanks of the head is also composed of 2d- and 4d-derived trunk tissue, thus corroborating the postulated descent of this region and its appendages from a cephalized anteriormost trunk segment and its parapodia. The cell-lineage domains of the first and third micromere tiers are arranged left or right of the sagittal plane, while two micromeres of the second quartet are in a lateral and, initially, two in a median position (2b ventral and 2d dorsal). The offspring of micromere 2d expand from a dorsal position toward the ventral midline and those of cell 4d from a posterior-dorsal site toward the anterior, initially forming two lateral bands. In the epispheric part of the head, part of the neurectodermal tissue derived from micromeres 1a and 1b interweaves in a medio-sagittal bar, and part of the first micromere offspring of all four quadrants (1a-1d) combine in forming a central brain neuropil. Each of the latter sends neurites through both of the circumesophageal connectives. Paired muscle tracts extend through the head toward the base of the antennae and are probably derived from micromeres 3a and 3b. A mesodermal envelope of the stomodeum is probably built by the 3c and 3d micromeres. The formation of symmetry and the nature of the body axes in the embryo and adult of Platynereis dumerilii are discussed. J. Morphol.