Pine embryogenesis: Many licences to kill for a new life

In plants, programmed cell death (PCD) is an important mechanism that controls normal growth and development as well as many defence responses. At present, research on PCD in different plant species is actively carried out due to the possibilities offered by modern methods in molecular biology and the increasing amount of genome data. The pine seed provides a favourable model for PCD because it represents an interesting inheritance of seed tissues as well as an anatomically well-described embryogenesis during which several tissues die via morphologically different PCD processes.Key words: conifer, developmental cell death, embryogenesis, megagametophyte, necrotic cell death, pine, seed development     

Predicting spring frost sensitivity by bud development and temperature sum in Norway spruce seedlings

Nurseries would benefit greatly if frost hardiness (FH) of seedlings could be predicted by some environmental variable or by bud development in spring. We investigated the FH of 1-year-old Norway spruce (Picea abies (L.) Karst.) seedlings of local origin. The seedlings were stored frozen until incubated in the growth chamber at six different temperature sums (TSs) (0, 55, 88, 142, 185 and 240 d.d., >5°C) from mid-February to mid-March. FH of the buds, stems and previous year needles was assessed on three occasions. When the TS was 88 d.d. or less, buds exhibited only microscopic signs of development, even when seedlings tolerated temperatures below −10°C. As TS increased, primordial needles and primordial stems of buds grew while FH weakened, especially in previous year needles. When the TS was at least 142 d.d., all plant parts were frost hardy to approximately −6°C. Monitoring TS and bud development can help predict FH of Norway spruce seedlings in spring. However, more studies with seedlings of different ages and from multiple locations are necessary to appreciate the generality of our results.     

Deficiency of a transmembrane prolyl 4-hydroxylase in the zebrafish leads to basement membrane defects and compromised kidney function

Prolyl 4-hydroxylases (P4Hs) catalyze the hydroxylation of collagens and hypoxia-inducible factor (HIF)-α subunits. We studied the zebrafish homologue of the recently characterized human transmembrane P4H (P4H-TM) that can hydroxylate HIF-α, but not collagens, in vitro and influence HIF-α levels in cellulo. The zebrafish P4H-TM mRNA had its highest expression in the eye and brain and lower levels in other tissues, including the kidney. Morpholino knockdown of P4H-TM in embryos resulted in a reduction in the size of the eye and head and morphological alterations in the head from 2 days postfertilization onward. In addition, pericardial edema, regarded as a sign of kidney dysfunction, developed from 3 days postfertilization onward. The phenotype was dependent on the P4H-TM catalytic activity because similar results were obtained with morpholinos targeting either translation initiation or catalytic residues of the enzyme. Structural and functional analyses of the morphant pronephric kidneys revealed fragmented glomerular basement membranes (BMs), disorganized podocyte foot processes, and severely compromised pronephric kidney function leading to proteinuria. The opacity of the eye lens was increased due to the presence of extra nuclei and deposits, and the structure of the lens capsule BM was altered. Our data suggest that P4H-TM catalytic activity is required for the proper development of the glomerular and lens capsule BMs. Many HIF target genes were induced in the P4H-TM-deficient morphants, but the observed phenotype is not likely to be mediated at least solely via the HIF pathway, and thus P4H-TM probably has additional, as yet unknown, substrates.     

Small intestinal permeability and serum folate and cobalamin absorption after surgical construction of permanent jejunal fistulas in laboratory beagle dogs

Permanent jejunal fistulas enable easy, noninjurious, repeated and direct administration to and collection from the small intestines of conscious laboratory dogs. This study aimed at identifying potential alterations in the small intestinal morphology and function of this canine model after the surgery required to establish the fistulas. Assays of serum folate and cobalamin and (51)Cr-EDTA permeability tests were performed before and 4 wk after experimental jejunoplasties in 14 laboratory beagle dogs. Serum folate concentrations (mean ± SD) before (12.22 ± 1.80 μg/L) and after (14.14 ± 1.70 μg/L) jejunal surgery were within reference ranges for healthy dogs, although folate concentrations were higher after surgery. The cobalamin concentrations and the 6-h urinary excretion of (51)Cr-EDTA before (573.50 ± 150.04 ng/L and 6.75 ± 1.56%, respectively) and after (496.71 ± 164.22 ng/L and 6.41 ± 1.10%) were normal for healthy dogs, and no significant differences between pre- and postsurgical values were detected. The findings of the present study indicate that the small intestinal vitamin absorption and permeability of laboratory beagle dogs with jejunal fistulas remains unimpaired.     

Quantitative real-time PCR for determination of microcystin synthetase e copy numbers for microcystis and anabaena in lakes

Cyanobacterial mass occurrences in freshwater lakes are generally formed by Anabaena, Microcystis, and Planktothrix, which may produce cyclic heptapeptide hepatotoxins, microcystins. Thus far, identification of the most potent microcystin producer in a lake has not been possible due to a lack of quantitative methods. The aim of this study was to identify the microcystin-producing genera and to determine the copy numbers of microcystin synthetase gene E (mcyE) in Lake Tuusulanj?rvi and Lake Hiidenvesi in Finland by quantitative real-time PCR. The microcystin concentrations and cyanobacterial cell densities of these lakes were also determined. The microcystin concentrations correlated positively with the sum of Microcystis and Anabaena mcyE copy numbers from both Lake Tuusulanj?rvi and Lake Hiidenvesi, indicating that mcyE gene copy numbers can be used as surrogates for hepatotoxic Microcystis and ANABAENA: The main microcystin producer in Lake Tuusulanj?rvi was Microcystis spp., since average Microcystis mcyE copy numbers were >30 times more abundant than those of ANABAENA: Lake Hiidenvesi seemed to contain both nontoxic and toxic Anabaena as well as toxic Microcystis strains. Identifying the most potent microcystin producer in a lake could be valuable for designing lake restoration strategies, among other uses.     

Further improvement of broad specificity hapten recognition with protein engineering

Sulfa-antibiotics (sulfonamides) are widely used in veterinary medicine. Meat and milk from treated animals can be contaminated with sulfa residues. Current sulfonamide assays are unfit for screening of food, because they are either too laborious, insensitive or specific for a few sulfa compounds only. An immunoassay for detection of all sulfas in a single reaction would be useful for screening. Previously we have improved the broad specificity sulfa binding of antibody 27G3 with random mutagenesis and phage display. In order to improve the properties of this antibody further, mutants from the previous study were recombined and more mutations introduced. These new libraries were enriched with phage display and several different mutant antibodies were isolated. The cross-reaction profile of the best mutant was better than that of the wild-type antibody and the mutants of the previous study: it was capable of binding 10 of the tested 13 sulfonamides within a narrow concentration range and also bound the rest of the sulfas 5- to 11-fold better than the mutants of the previous study.     

Constitutive precoupling to G(i) and increased agonist potency in the alpha(2B)-adrenoceptor

The human alpha(2B)-adrenoceptor (alpha(2B)-AR) was mutated by substituting the D(3.49) aspartate in position 109 with an alanine (alpha(2B)-D109A) in the conserved DRY sequence at the cytoplasmic face of TM3. We studied the effects of the mutation on agonist binding and on receptor activation in CHO cells, including possible inverse agonism monitored by measuring intracellular Ca(2+) concentrations ([Ca(2+)](i)). The mutated receptor had increased binding affinity for agonists, especially dexmedetomidine (3.8-fold). The increased affinity was abolished by pretreatment of the cells with pertussis toxin. The mutation produced constitutive receptor activity evidenced as increased basal [Ca(2+)](i) and increased potency and efficacy of agonists to elicit Ca(2+) responses. The imidazoline derivative RX821002 functioned as an inverse agonist only through the alpha(2B)-D109A, reducing [Ca(2+)](i). The results thus indicate that this mutation causes constitutive receptor-G(i)-protein precoupling, and that the D(3.49) aspartate residue of the DRY motif is involved in controlling coupled and uncoupled conformations of alpha(2B)-AR.     

alpha 2B-Adrenoceptor levels govern agonist and inverse agonist responses in PC12 cells

Receptor density is an important determinant of cellular effector responses to receptor activation. We analysed cytosolic Ca(2+) responses to alpha(2)-adrenergic agents in PC12 cells expressing human alpha(2B)-adrenergic receptors (AR) at two densities (3.8 and 1.3 pmol/mg protein). The efficacy (E(max)) of agonists was greater in cells with higher receptor expression; while the potency (EC(50)) of norepinephrine and oxymetazoline was independent of alpha(2B)-AR levels. Several classical alpha(2)-AR antagonists behaved as either partial or inverse agonists in a receptor density-dependent fashion. No apparent structural similarities were found among the inverse agonists, precluding simple predictions of inverse agonist activity. Transfected PC12 cells expressing alpha(2B)-AR at relatively high density would be a useful approach to screen inverse agonists for this class of receptors. Our results further indicate that receptor density significantly influences the properties of ligands, not only of partial agonists as predicted by classical receptor theory, but also of antagonists and full agonists.     

Methodologies for the characterization of microbes in industrial environments: a review

There is growing interest in research and development to develop novel tools to study, detect, and characterize microbes and their communities in industrial environments. However, knowledge about their validity in practical industrial use is still scarce. This review describes the advantages and limitations of traditional and molecular methods used for biofilm and/or planktonic cell studies, especially those performed with Listeria monocytogenes, Bacillus cereus, and/or Clostridium perfringens. In addition, the review addresses the importance of isolating the microorganisms from the industrial environment and the possibilities and future prospects for exploiting the described methods in the industrial environment.     

Phosphorylation of a peptide related to subunit c of the F0F1-ATPase/ATP synthase and relationship to permeability transition pore opening in mitochondria

A phosphorylated polypeptide (ScIRP) from the inner membrane of rat liver mitochondria with an apparent molecular mass of 3.5 kDa was found to be immunoreactive with specific antibodies against subunit c of F₀F₁-ATPase/ATP synthase (Azarashvily, T. S., Tyynelä, J., Baumann, M., Evtodienko, Yu. V., and Saris, N.-E. L. (2000). Biochem. Biophys. Res. Commun. 270, 741–744. In the present paper we show that the dephosphorylation of ScIRP was promoted by the Ca²⁺-induced mitochondrial permeability transition (MPT) and prevented by cyclosporin A. Preincubation of ScIRP isolated in its dephosphorylated form with the mitochondrial suspension decreased the membrane potential (_M) and the Ca²⁺-uptake capacity by promoting MPT. Incorporation of ScIRP into black-lipid membranes increased the membrane conductivity by inducing channel formation that was also suppressed by antibodies to subunit c. These data indicate that the phosphorylation level of ScIRP is influenced by the MPT pore state, presumably by stimulation of calcineurin phosphatase by the Ca²⁺ used to induce MPT. The possibility of ScIRP being part of the MPT pore assembly is discussed in view of its capability to induced channel activity.