Cell lineages in the embryonic kidney: their inductive interactions and signalling molecules.

The first signalling genes acting in the inductive interactions in the kidney have now been identified. Differentiation of the permanent kidney or the metanephros is critically dependent on inductive signalling between the nephrogenic mesenchyme and ureteric bud epithelium. Further inductive interactions occur between developing nephrons, interstitial stroma, endothelial cells and neurones. Glial-cell-line-derived neurotrophic factor is a signal for the ureteric bud initiation and branching, and Wnt4 is an autocrine epithelializing signal at the pretubular stage of nephron formation. The signals for renal angiogenesis and innervation are less well defined, but seem to include vascular endothelial growth factor and neurotrophins, at least. The ureteric-bud-derived signal for induction of the nephrogenic mesenchyme (to bring the cells to the condensate stage) is not yet known, but fibroblast growth factor 2 is a good candidate. None of the signalling genes identified from the embryonic kidney is specific to the organ, which raises some general questions. How do the organs develop from similar rudiments to various patterns with different cell types and functions? Does the information for organ-specific differentiation pathways retain in the epithelial or mesenchymal compartment? The present, rather fragmentary molecular data would favour the view that similar molecules acting in different combinations and developmental sequences, rather than few organ-specific master genes, could be responsible for the divergence of patterning.     

Regio and stereospecific DNA adduct formation in mouse lung at N6 and N7 position of adenine and guanine after 1,3 butadiene inhalation exposure

Butadiene monoepoxide (BMO) alkylated guanine N7 and adenine N 6 adducts were prepared and enriched by solid phase extraction and HPLC. The purified adducts were analysed by a modified 32P-postlabelling assay, which utilized one dimensional TLC chromatography and a subsequent HPLC analysis with UV and radioactivity detectors. In vitro with Ct-DNA the formation of N7-dGMP and N 6-dAMP adducts were linear at a concentration range of 44 to 870 nmol of BMO per mg DNA at physiological pH. N7- dGMP and N 6-dAMP adducts were formed in a ratio of 200:1. In dGMP and in dAMP 48 % and 86 % of adducts were covalently bound to the C-2 carbon of BMO. CD-1 mice were inhalation exposed to butadiene for 5 days and 6 h per day. The N7-dGMP adduct level in lung samples of animals exposed to 200, 500 and 1300 ppm was 2.8 +/- 0.9 fmol, 11 +/- 2.0 fmol and 30 +/- 6.7 fmol in 10 mug DNA, respectively. The level of N 6-dAMP adducts in lung samples after 500 ppm and 1300 ppm exposure was 0.09 +/- 0.06 fmol and 0.11 +/- 0.05 fmol in 10 mug DNA. At 200 ppm the adduct level was below the detection limit. A sub-group of animals exposed to 1300 ppm was killed 3 weeks after the last exposure. N7-dGMP adducts were not detected but the level of N 6-dAMP adducts was not affected. N7-dGMP adducts were formed in a clear stereospecific manner in vivo . S -BMO adducts were the main product and represented 77 % ( n = 4, SD = 2%) of total BMO adducts. No clear conclusion can be drawn about the enantiospecific DNA binding at the N 6 position of dAMP, because of the poor separation of the enantiomers. However, we could separate regioisomeric adducts which indicated that C-2 adducts represented 69 +/- 3 % of the total N 6 adducts formed in mice lung DNA. This observation is supported by the data derived from in vitro DNA experiments but is different to our previously published data, which indicates the 2:1 (C-1:C-2) ratio in regioisomer formation in nucleotides or nucleosides. We suggest that the data presented in this communication indicate a different mechanism between nucleotides and DNA in BMO-derived adduct formation- Dimroth rearrangement dominates in nucleotides, but in double stranded DNA a direct alkylation is probably the major mechanism of adduct formation.     

Secretory organelles in ECL cells of the rat stomach: an immunohistochemical and electron-microscopic study

ECL cells are numerous in the rat stomach. They produce and store histamine and chromogranin-A (CGA)-derived peptides such as pancreastatin and respond to gastrin with secretion of these products. Numerous electron-lucent vesicles of varying size and a few small, dense-cored granules are found in the cytoplasm. Using confocal and electron microscopy, we examined these organelles and their metamorphosis as they underwent intracellular transport from the Golgi area to the cell periphery. ECL-cell histamine was found to occur in both cytosol and secretory vesicles. Histidine decarboxylase, the histamine-forming enzyme, was in the cytosol, while pancreastatin (and possibly other peptide products) was confined to the dense cores of granules and secretory vesicles. Dense-cored granules and small, clear microvesicles were more numerous in the Golgi area than in the docking zone, i.e. close to the plasma membrane. Secretory vesicles were numerous in both Golgi area and docking zone, where they were sometimes seen to be attached to the plasma membrane. Upon acute gastrin stimulation, histamine was mobilized and the compartment size (volume density) of secretory vesicles in the docking zone was decreased, while the compartment size of microvesicles was increased. Based on these findings, we propose the following life cycle of secretory organelles in ECL cells: small, electron-lucent microvesicles (pro-granules) bud off the trans Golgi network, carrying proteins and secretory peptide precursors (such as CGA and an anticipated prohormone). They are transformed into dense-cored granules (approximate profile diameter 100 nm) while still in the trans Golgi area. Pro-granules and granules accumulate histamine, which leads to their metamorphosis into dense-cored secretory vesicles. In the Golgi area the secretory vesicles have an approximate profile diameter of 150 nm. By the time they reach their destination in the docking zone, their profile diameter is between 200 and 500 nm. Exocytosis is coupled with endocytosis (membrane retrieval), and microvesicles in the docking zone are likely to represent membrane retrieval vesicles (endocytotic vesicles).     

Linkage of aspartylglucosaminuria (AGU) to marker loci on the long arm of chromosome 4

Summary Aspartylglucosaminuria (AGU) is caused by deficient activity of the enzyme aspartylglucosaminidase (AGA). The structural gene for AGA has been assigned to the region 4q21-qter of chromosome 4. We have studied the map position of the AGU locus in relation to other marker loci on the long arm of chromosome 4 using linkage analyses. Restriction fragment length polymorphism alleles for the ADH2, ADH3, EGF, FG and FG loci and blood group antigenes for the MNS locus were determined in a panel of 12 Finnish AGU families. The heterozygous family members were identified by reduced activity of AGA in lymphocytes. Linkage studies were performed using both pairwise and multipoint analyses. Loose linkage of the AGU locus to the FG and MNS loci was observed ( = 1.16, = 1.39, respectively). Multipoint analysis to the fixed map [ADH-(0.03)-EGF-(0.35)-FG-(0.11)-MNS] suggests that the location of the AGU locus is 0.05–0.30 recombination units distal to MNS ( = 3.03). The order cen-ADH-EGF-FG-MNS-AGU is 35 times more likely than the next best order cen-ADH-EGF-AGU-FG-MNS.     

Dynamic states of the DNA repair enzyme AlkB regulate product release

The 2-oxoglutarate (2OG)- and Fe(2+)-dependent dioxygenase AlkB couples the demethylation of modified DNA to the decarboxylation of 2OG. Extensive crystallographic analyses have shown no evidence of significant structural differences between complexes binding either 2OG or succinate. By using nuclear magnetic resonance spectroscopy, we have shown that the AlkB-succinate and AlkB-2OG complexes have significantly different dynamic properties in solution. 2OG makes the necessary contacts between the metal site and the large beta-sheet to maintain a fully folded conformation. Oxidative decarboxylation of 2OG to succinate leads to weakening of a main contact with the large beta-sheet, resulting in an enhanced dynamic state. These conformational fluctuations allow for the replacement of succinate in the central core of the protein and probably contribute to the effective release of unmethylated DNA. We also propose that the inherent dynamics of the co-product complex and the subsequent increased molecular ordering of the co-substrate complex have a role in DNA damage recognition.     

Deletion of lrrk2 causes early developmental abnormalities and age-dependent increase of monoamine catabolism in the zebrafish brain

LRRK2 gain-of-function is considered a major cause of Parkinson’s disease (PD) in humans. However, pathogenicity of LRRK2 loss-of-function in animal models is controversial. Here we show that deletion of the entire zebrafish lrrk2 locus elicits a pleomorphic transient brain phenotype in maternal-zygotic mutant embryos (mzLrrk2). In contrast to lrrk2, the paralog gene lrrk1 is virtually not expressed in the brain of both wild-type and mzLrrk2 fish at different developmental stages. Notably, we found reduced catecholaminergic neurons, the main target of PD, in specific cell populations in the brains of mzLrrk2 larvae, but not adult fish. Strikingly, age-dependent accumulation of monoamine oxidase (MAO)-dependent catabolic signatures within mzLrrk2 brains revealed a previously undescribed interaction between LRRK2 and MAO biological activities. Our results highlight mzLrrk2 zebrafish as a tractable tool to study LRRK2 loss-of-function in vivo, and suggest a link between LRRK2 and MAO, potentially of relevance in the prodromic stages of PD.     

Temperature dependence of leaf-level CO2 fixation: revising biochemical coefficients through analysis of leaf three-dimensional structure

CO2 fixation in a leaf is determined by biochemical and physical processes within the boundaries set by leaf structure. Traditionally determined temperature dependencies of biochemical processes include physical processes related to CO2 exchange that result in inaccurate estimates of parameter values. A realistic three-dimensional model of a birch (Betula pendula) leaf was used to distinguish between the physical and biochemical processes affecting the temperature dependence of CO2 exchange, to determine new chloroplastic temperature dependencies for V c(max) and Jmax based on experiments, and to analyse mesophyll diffusion in detail. The constraint created by dissolution of CO2 at cell surfaces substantially decreased the CO2 flux and its concentration inside chloroplasts, especially at high temperatures. Consequently, newly determined chloroplastic V c(max) and Jmax were more temperature dependent than originally. The role of carbonic anhydrase in mesophyll diffusion appeared to be minor under representative mid-day nonwater-limited conditions. Leaf structure and physical processes significantly affect the apparent temperature dependence of CO2 exchange, especially at optimal high temperatures when the photosynthetic sink is strong. The influence of three-dimensional leaf structure on the light environment inside a leaf is marked and affects the local choice between Jmax and V c(max)-limited assimilation rates.     

Carbon dioxide and methane fluxes in boreal peatland microcosms with different vegetation cover—effects of ozone or ultraviolet-B exposure

O₃ concentrations in the troposphere are rising and those in the stratosphere decreasing, the latter resulting in higher fluxes of solar ultraviolet-B (UV-B) radiation to the earth's surface. We assessed whether the fluxes of CO₂ and CH₄ are altered by enhanced UV-B radiation or elevated tropospheric O₃ concentrations in boreal peatland microcosms (core depth 40 cm, diameter 10.5 cm) with different vegetation cover. At the end of the UV-B experiment which lasted for a growing season, net CO₂ exchange (NEE) and dark ecosystem respiration (R _TOT) were sevenfold higher, and CH₄ efflux 12-fold higher, in microcosms with intact vegetation dominated by Eriophorum vaginatum L. and Sphagnum spp., compared to microcosms from which we removed E. vaginatum. Vegetation treatment had minor effects on CH₄ production and consumption potentials in the peat, suggesting that the large difference in CH₄ efflux is mainly due to efficient CH₄ transport via the aerenchyma of E. vaginatum. Ambient UV-B supplemented with 30% and elevated O₃ concentrations (100 and 200 ppb, for 7 weeks) significantly increased R _TOT in both vegetation treatments. Elevated O₃ concentrations reduced NEE over time, while UV-B had no clear effects on the fluxes of CO₂ or CH₄ in the cloudy summer of the study. Field experiments are needed to assess the significance of increasing UV-B radiation and elevated tropospheric O₃ concentration on peatland gas exchange in the long-term.     

Prevention and suppression of Helicobacter felis infection in mice using colostral preparation with specific antibodies

Background. Specific antibodies against Helicobacter were enriched from the colostra of hyperimmunized cows. Efficacies of colostral control preparation and immune preparation containing specific antibodies against Helicobacter felis were studied in the prevention and treatment of experimental H. felis infection in mice. Materials and Methods. H. felis‐infected mice were given either immune or control preparation with or without complement or amoxicillin orally in four different trials. H. felis status was assessed on the basis of bacterial stainings, gastric histology and serum antibodies. Results. Immune, but not control preparation, prevented H. felis infection (p > 0.01), the efficacy being dependent on the presence of specific antibodies. In the trial on infected Balb/c mice treatment with immune preparation (p = 0.029) but not control preparation decreased the colonization of gastric antrum by H. felis. In the further trials with infected SJL‐mice, treatments with colostral preparations did not decrease colonization. Amoxicillin treatment decreased the colonization with trend‐setting significance (p = 0.056; infected mice as controls), whereas amoxicillin combined with immune preparation had a significant effect (p < 0.0005). Conclusions. Specific colostral antibodies were useful in the prevention of Helicobacter infection in a mouse model. The results of the treatment trials were controversial but a similar colostral immune preparation against H. pylori could be effective and useful in preventing infections in humans and during antibiotic treatment.     

Rapid extraction of phycobiliproteins from cultured cyanobacteria samples

Cyanobacteria are a valuable and ubiquitous component of marine picophytoplankton that contribute significantly to total carbon biomass and primary productivity of the oceans. They contain water soluble, natively highly fluorescent proteins, phycobiliproteins, that can be considered ideal marker pigments for understanding the distribution and trophic dynamics of picoplankton populations. However, there is no standard protocol for extracting and quantitating these proteins from cyanobacterial cells. Ideally, the cells would be disrupted quickly and efficiently with complete extraction and recovery of the released proteins. For that purpose, we describe a method for extracting phycobiliproteins from a Synechococcus CCMP 833 cyanobacteria culture that utilizes 3% 3-[(3-cholamidopropyl)dimethyammonio]propanesulfonic acid (Chaps) 0.3% asolectin combined with nitrogen cavitation. Extraction efficiencies of greater than 85% were achieved by this method, which requires less than 3h. The analysis of the extracted samples was carried out by capillary electrophoresis with laser-induced fluorescence detection.