[Lobectomy of Right Lower Lobe Eight Years after Foreign Body Aspiration]. / Unterlappenektomie acht Jahre nach Aspiration einer Pinnnadel.

A young man presented with haemoptysis, eight years after foreign body aspiration. The initial evaluation took place in the emergency department of a general hospital. However, neither chest x-ray nor bronchoscopy were performed. Bronchoscopy performed in our hospital revealed a foreign body in right lower lobe bronchus. Extraction failed because it was embedded in granulation tissue. The chronic atelectasis of right lower lobe and recurrent bronchopulmonary infections during the last years were the indication for lobectomy.

Developmentally regulated expression of a cyclic nucleotide-gated ion channel from Arabidopsis indicates its involvement in programmed cell death.

Cyclic nucleotide-gated ion channels have been identified in animals and plants. However, the physiological role of these ion channels in plant cells and in non-receptor cells of animals is still unknown. Here, we focused on one member of the large gene family of cyclic nucleotide-gated ion channels from Arabidopsis thaliana (L.) Heynh., AtCNGC2. The analysis of the transcriptional regulation revealed that expression of AtCNGC2 is low in etiolated seedlings but increases substantially during de etiolation. The use of promoter::GUS plants revealed that expression of AtCNGC2 in seedlings is highest in cotyledons after release of the developmental arrest by light. Expression of AtCNGC2 was also observed in later stages of plant development. Investigations using the promoter::GUS plants demonstrated that AtCNGC2 is expressed in flowers during organ senescence and in the dehiscence zone of siliques. Furthermore, expression of AtCNGC2 was transiently induced during leaf and cell culture senescence. These results indicate a potential function for AtCNGC2 in the initiation of developmentally regulated cell death programs.

Relevance of low titers of cryoglobulins and cold-agglutinins in patients with isolated Raynaud phenomenon.

INTRODUCTION: A Raynaud phenomenon can be associated with cold agglutinins or cryoglobulins. Although cold agglutinins or cryoglobulins may lead to severe acral gangrene the finding of relevant titers is rare. Low titers of cold agglutinins or cryoglobulins are detected more frequently but are assumed to be without any importance. OBJECTIVES: To prove a possible diagnostic or prognostic role of low titers of cold agglutinins or cryoglobulins in patients presenting an isolated Raynaud phenomenon we did a retrospective analysis. SETTINGS AND SUBJECTS: In 306 patients (40+/-16 years, range: 15-83 years) with a mean duration of the history of an isolated Raynaud phenomenon of 48+/-73 months we did a clinical examination, an analysis of antinuclear antibodies, extractable antibodies, cold agglutinins, cryoglobulins, plasma and blood viscosity, erythrocyte aggregation and a nail fold capillaroscopy. RESULTS: Low titers of cold agglutinins were found in 49 patients and of cryoglobulins in 7 patients. The finding of such low titers was not associated with extensive clinical symptoms, duration of clinical symptoms, megacapillaries or haemorrhagies in capillaroscopy, pathologic plasma and blood viscosity and erythrocyte aggregation. The follow-up investigations (mean: 3.1+/-1.2 years, range: 3-7 years) revealed no development of a haematological, vasculitic or connective tissue disease in the subgroup of patients who only had low titers of cold agglutinins. CONCLUSION: The detection of low titers of cold agglutinins in patients with isolated Raynaud phenomenon is of no diagnostic or prognostic relevance.

Auxin distribution and transport during embryonic pattern formation in wheat.

Inhibitors of auxin polar transport disrupt normal embryogenesis and thus specific spatial auxin distribution due to auxin movement may be important in establishing embryonic pattern formation in plants. In the present study, the distribution of the photoaffinity labeling agent tritiated 5-azidoindole-3-acetic acid ([3H],5-N3IAA), an analog of indole-3-acetic acid (IAA), was visualized in zygotic wheat (Triticum aestivum L.) embryos grown in vitro and in planta, and used to deduce auxin transport pathways in these embryos. This study provides the first direct evidence that the distribution of auxin, here [3H],5-N3IAA, is heterogeneous and changes during embryo development. In particular, the shift from radial to bilateral symmetry was correlated with a redistribution of [3H],5-N3IAA in the embryo. Furthermore, in bilaterally symmetrical embryos, that is, embryos in the late transition stage or older, the localization of [3H],5-N3IAA was altered by N-1-naphthylphthalamic acid, a specific inhibitor of auxin polar transport. No significant effect was observed in radially symmetrical embryos, that is, globular embryos, or very early transition embryos. Thus, the shift from radial to bilateral symmetry is associated with the onset of active, directed auxin transport involved in auxin redistribution. A change in the distribution of [3H],5-N3IAA was also observed in morphologically abnormal embryos induced on media supplemented with auxin or auxin polar transport inhibitors. By means of a microscale technique, free IAA concentration was measured in in vitro- and in planta-grown embryos and was found to increase during development. Therefore, IAA may be synthesized or released from conjugates in bilaterally symmetrical embryos, although import from surrounding tissues cannot be excluded.

A novel phytyltransferase from Synechocystis sp. PCC 6803 involved in tocopherol biosynthesis.

The deduced polypeptide sequence of open reading frame slr1736 reveals homology to chlorophyll synthase and 1,4-dihydroxy-2-naphthoic acid phytyltransferase in Synechocystis sp. strain PCC 6803. In tocopherol and plastoquinone biosynthesis, a condensation reaction mechanistically similar to that of these two enzymes is performed. To analyze the function of this novel prenyltransferase, a deletion mutant of slr1736 was generated by homologous recombination. The mutant showed a markedly decreased tocopherol content, while plastoquinone levels remained unchanged. Since the aromatic precursor homogentisic acid accumulated in the mutant, the function of the enzyme was proven to be a novel tocopherol phytyltransferase.

Measures of information processing in rapid automatized naming (RAN) and their relation to reading.

The letters, numbers, and objects subtests of the Rapid Automatized Naming Tests (RAN) were given to 50 first- and second-grade students. Student performance on the three RAN subtests were audiotaped and subjected to postacquisition processing to distinguish articulation and interarticulation pause times. This study investigated (1) the relations between the articulation and pause durations associated with the 50 stimuli of each RAN subtest and (2) the relations between the pause and articulation latencies of the three RAN subtests and reading. For both first- and second-grade students, pause and articulation times for RAN letters and objects were not found to be reliably related, in contrast to RAN numbers articulation and pause durations. RAN subtest pause durations were differentially related to reading; however, articulation was rarely related to reading. The RAN letters pause time was the most robust predictor of decoding and reading comprehension, consistently predicting all first- and second-grade measures. Analysis supported the view that reading is predicted by speed of processing associated with letters, not general processing speed.

Plant heterotrimeric G protein beta subunit is associated with membranes via protein interactions involving coiled-coil formation.

Gbeta subunits from animals are anchored to membranes via Ggamma subunits. No Ggamma has been identified in plants to date. Using differential centrifugation of Arabidopsis and broccoli extracts, Gbeta was highly enriched in the microsomal pellet. Treatment of microsomes with detergents and salts indicates that plant Gbeta is located at the membrane surface and attached to membranes by hydrophobic interactions. Analysis of transgenic plants expressing Gbeta-GFP fusion proteins showed that mutations in the heptad repeat domain of Gbeta severely diminished their membrane association. We propose that plant Gbeta is anchored to membranes by an unknown protein similar to animal Gbeta by Ggamma, via coiled-coil formation.

Interleukin-6 stimulates LDL receptor gene expression via activation of sterol-responsive and Sp1 binding elements.

Inflammatory or malignant diseases are associated with elevated levels of cytokines and abnormal low density lipoprotein (LDL) cholesterol metabolism. In the acute-phase response to myocardial injury or other trauma or surgery, total and LDL cholesterol levels are markedly decreased. We investigated the effects of the proinflammatory cytokine interleukin (IL)-6 on LDL receptor (LDL-R) function and gene expression in HepG2 cells. IL-6 dose-dependently increased the binding, internalization, and degradation of (125)I-LDL. IL-6-stimulated HepG2 cells revealed increased steady-state levels of LDL-R mRNA. In HepG2 cells transiently transfected with reporter gene constructs harboring the sequence of the LDL-R promoter extending from nucleotide -1563 (or from nucleotide -234) through -58 relative to the translation start site, IL-6 dose-dependently increased promoter activity. In the presence of LDL, a similar relative stimulatory effect of IL-6 was observed. Studies using a reporter plasmid with a functionally disrupted sterol-responsive element (SRE)-1 revealed a reduced stimulatory response to IL-6. In gel-shift assays, nuclear extracts of IL-6-treated HepG2 cells showed an induced binding of SRE binding protein (SREBP)-1a and SRE binding protein(SREBP)-2 to the SRE-1 that was independent of the cellular sterol content and an induced binding of Sp1 and Sp3 to repeat 3 of the LDL-R promoter. Our data indicate that IL-6 induces stimulation of the LDL-R gene, resulting in enhanced gene transcription and LDL-R activity. This effect is sterol independent and involves, on the molecular level, activation of nuclear factors binding to SRE-1 and the Sp1 binding site in repeat 2 and repeat 3 of the LDL-R promoter, respectively.

Characterisation of calmodulin binding to cyclic nucleotide-gated ion channels from Arabidopsis thaliana.

The recently identified cyclic nucleotide-gated ion channels (AtCNGCs) from Arabidopsis thaliana have the ability to bind calmodulin. Using two different methods, we mapped the binding site of AtCNGC1 to the last predicted alpha helix of the cyclic nucleotide binding domain. This is in contrast to CNGCs from animals, where the calmodulin binding site is located in the N-terminus, implying that different mechanisms for CNGC modulation have evolved in animals and plants. Furthermore, we demonstrate that AtCNGC1 and AtCNGC2 have different calmodulin binding affinities and we provide evidence for target specificities among calmodulin isoforms.

Characterisation of a novel gene family of putative cyclic nucleotide- and calmodulin-regulated ion channels in Arabidopsis thaliana.

In plants, cyclic GMP is involved in signal transduction in response to light and gibberellic acid. For cyclic AMP, a potential role during the plant cell cycle was recently reported. However, cellular targets for cyclic nucleotides in plants are largely unknown. Here we report on the identification and characterisation of a new gene family in Arabidopsis, which share features with cyclic nucleotide-gated channels from animals and inward-rectifying K+ channels from plants. The identified gene family comprises six members (Arabidopsis thaliana cyclic nucleotide-gated channels, AtCNGC1-6) with significant homology among the deduced proteins. Hydrophobicity analysis predicted six membrane-spanning domains flanked by hydrophilic amino and carboxy termini. A putative cyclic nucleotide binding domain (CNBD) which contains several residues that are invariant in other CNBDs was located in the carboxy terminus. This domain overlaps with a predicted calmodulin (CaM) binding site, suggesting interaction between cyclic nucleotide and CaM regulation. We demonstrated interaction of the carboxy termini of AtCNGC1 and AtCNGC2 with CaM in yeast, indicating that the CaM binding sites are functional. Furthermore, it was shown that both AtCNGC1 and AtCNGC2 can partly complement the K(+)-uptake-deficient yeast mutant CY162. Therefore, we propose that the identified genes constitute a family of plant cyclic nucleotide- and CaM-regulated ion channels.

Nuclear export of proteins in plants: AtXPO1 is the export receptor for leucine-rich nuclear export signals in Arabidopsis thaliana.

Transport across the nuclear envelope is mediated by transport receptors from the Importin beta family. We identified Exportin 1 from Arabidopsis (AtXPO1/AtCRM1) as the nuclear export receptor for proteins carrying leucine-rich nuclear export signals (NESs). AtXPO1 shares 42-50% identity with its functional homologues from humans and yeasts. We functionally characterised AtXPO1 by its interaction with NESs of animal and plant proteins, which is inhibited by the cytotoxin leptomycin B (LMB), and also by its interaction with the small GTPase Ran1 in the yeast two-hybrid system. Furthermore, we demonstrated the existence of a nuclear export pathway for proteins in plants. For the characterisation of nuclear export activities, we established an in vivo assay based on the localisation equilibrium of a GFP reporter protein fused to both a nuclear localisation signal (NLS) and an NES motif. Using this in vivo assay we demonstrated that the NES of the heterologous protein Rev is also functional in plants and that its export is inhibited by LMB. In addition, we identified a leucine-rich NES in the Arabidopsis protein AtRanBP1a. The NES, which is located at the carboxy terminus of the protein, is disrupted by mutating three long chain hydrophobic amino acid residues to alanine (L176A, L179A, V181A). In BY-2 protoplasts the NES of AtRanBP1a is functionally indistinguishable from the Rev NES. Our results demonstrate that the machinery for the nuclear export of proteins is functionally conserved in plants.

Auxin-induced developmental patterns in Brassica juncea embryos.

To investigate the mechanism of auxin action during pattern formation in dicot embryos, we tested the effects of the natural auxin indole-3-acetic acid (IAA), the auxin transport inhibitor N-(1-naphthyl)thalamic acid (NPA) and the antiauxin p-chlorophenoxyisobutyric acid (PCIB). In vitro treatments of isolated zygotic Brassica juncea embryos with these substances led to a wide range of morphogenetic alterations. Treatment of globular embryos with exogenous auxin (10-40 microM) either completely inhibited morphogenesis, resulting in ball-shaped embryos, or caused the development of egg- and cucumber-shaped embryos, which only consisted of a shortened hypocotyl without any apical structures. Axis duplication was observed sometimes after inhibition of auxin transport in globular embryos, and led to the development of twin embryos. During the transition from globular to heart stage, changes in auxin distribution or activity frequently caused the development of either split-collar or collar-cotyledons. Antiauxin inhibited cotyledon growth, leading to embryos with single or no cotyledons, or inhibited the development of the hypocotyl and the radicle. Inhibition of auxin transport in transition embryos sometimes led to axis broadening, which resulted in the development of two radicles. The described changes in embryo shapes represent arrests in different auxin-regulated developmental steps and phenocopy some Arabidopsis morphogenetic mutants.

Permutational distribution of the log-rank statistic under random censorship with applications to carcinogenicity assays.

In the random censorship model, the log-rank test is often used for comparing a control group with different dose groups. If the number of tumors is small, so-called exact methods are often applied for computing critical values from a permutational distribution. Two of these exact methods are discussed and shown to be incorrect. The correct permutational distribution is derived and studied with respect to its behavior under unequal censoring in the light of recent results proving that the permutational version and the unconditional version of the log-rank test are asymptotically equivalent even under unequal censoring. The log-rank test is studied by simulations of a realistic scenario from a bioassay with small numbers of tumors.

Phytochrome-regulated repression of gene expression requires calcium and cGMP.

The plant photoreceptor phytochrome A utilizes three signal transduction pathways, dependent upon calcium and/or cGMP, to activate genes in the light. In this report, we have studied the phytochrome A regulation of a gene that is down-regulated by light, asparagine synthetase (AS1). We show that AS1 is expressed in the dark and repressed in the light. Repression of AS1 in the light is likely controlled by the same calcium/cGMP-dependent pathway that is used to activate other light responses. The use of the same signal transduction pathway for both activating and repressing different responses provides an interesting mechanism for phytochrome action. Using complementary loss- and gain-of-function experiments we have identified a 17 bp cis-element within the AS1 promoter that is both necessary and sufficient for this regulation. This sequence is likely to be the target for a highly conserved phytochrome-generated repressor whose activity is regulated by both calcium and cGMP.

Induction of Zygotic Polyembryos in Wheat: Influence of Auxin Polar Transport.

The effects of two auxin polar transport inhibitors, N-1-naphthylphthalamic acid (NPA) and 3,3[prime],4[prime],5,7-pentahydroxyflavone (quercetin), on attaining bilateral symmetry from radial symmetry during early wheat embryogenesis were investigated by using an in vitro culture system. Although NPA and quercetin belong to two different classes of auxin transport inhibitors, the phytotropins and the flavonoids, respectively, they induced the same specific abnormal phenotypes during embryo development. These abnormal embryos differentiated multiple meristems (i.e., multiple shoot and root meristems) and multiple organs (i.e., multiple coleoptiles and scutella). Multiple shoot apical meristem phenotypes were characterized by partly multiplied embryonic axes and supernumerary scutella. The differentiation of multiple primary roots in addition to multiple shoot meristems and multiple scutella led to the formation of polyembryos. The occurrence of multiple shoot meristem phenotypes depended on the concentration of the inhibitor and the developmental stage of the isolated embryo. Embryos treated with NPA or quercetin developed multiple radicle phenotypes less frequently than they developed multiple shoot meristem phenotypes. Our observations suggest that the root meristem differentiates later than the shoot meristem. Our data support the hypothesis that polar transport of auxin has a determining influence on the differentiation of the embryonic axis and the scutellum.

Calcium and cGMP target distinct phytochrome-responsive elements.

Previous work using microinjection into single cells of the tomato aurea mutant demonstrated that phytochrome A-dependent activation of rbcS and chs genes was mediated by calcium and cGMP, respectively. This work sought to identify promoter cis-elements that respond to these two small molecules. Box II and Unit I, derived from rbcS-3A and chs promoters, respectively, were previously shown to function as light-responsive cis-elements. Eleven copies of Box II and four copies of Unit I were linked 5' to the -90 and -46 35 S promoters, respectively, and, both constructs were fused to the beta-glucuronidase (GUS) reporter gene. GUS activities were obtained upon coinjection of either Box II/-90GUS or Unit I/-46GUS with oat phytochrome A (phyA) and GTP gamma S, an activator of heterotrimeric G proteins. The activation of Box II/-90GUS by phyA was insensitive to the cGMP antagonist, Rp-cGMPS, although anthocyanin accumulation, but not chloroplast development, was totally blocked in the injected cells. Consistent with this result, calcium, but not cGMP, induced Box II/-90GUS activity. In contrast to Box II/-90GUS, phyA-dependent activation of Unit I/-46GUS activity was blocked by Rp-cGMPS. Moreover, cGMP, not calcium, induced Unit I/-46GUS activity. Control experiments showed that -90 GUS and -46 GUS were inactive in the presence of calcium and cGMP, respectively. These results provide evidence that Box II and Unit I are targets of the calcium and cGMP pathways, respectively. Interestingly, calcium activation of Box II/-90GUS was repressed by a high concentration of cGMP and cGMP induction of Unit I/-46GUS was blocked by a high concentration of calcium/CaM. Thus, these two small cis-elements can also serve as targets of the reciprocal control mechanisms that operate to regulate the activities of the two phyA signaling branches.

Functional analysis of yeast-derived phytochrome A and B phycocyanobilin adducts.

Investigations of phytochrome mutants of Arabidopsis suggested that the expression of chalcone synthase (chs) and anthocyanin accumulation is predominantly controlled by phytochrome A. To test the functionality of phytochrome A and B at the molecular level recombinant, yeast-derived phytochrome-phycocyanobilin adducts (phyA, phyB) and oat phytochrome A (phyA) were microinjected into etiolated aurea tomato seedlings. Subsequent to microinjection anthocyanin and chlorophyll accumulation was monitored as well as beta-glucuronidase (GUS) expression mediated by light-regulated promoters (chs, chlorophyll a/b binding protein (lhcb1) and ferredoxin NADP+ oxidoreductase (fnn). Microinjection of phyA under white light conditions caused anthocyanin and chlorophyll accumulation and mediated chs-GUS, lhcb 1-GUS and fnr-GUS expression. Microinjection of phyB under identical conditions induced chlorophyll accumulation and mediated lhcb 1-GUS and fnr-GUS expression but neither anthocyanin accumulation nor chs-GUS expression were observed. The characterization of Arabidopsis phytochrome mutants and the microinjection experiments suggested that phyB cannot induce the accumulation of juvenile anthocyanin. Microinjections under far-red light conditions demonstrated that phyA can act independently of other photoreceptors. By contrast, phyB injections under red light conditions indicated that phyB needs interactions with other photoreceptors to mediate a rapid and efficient de-etiolation signal.

Cholera toxin elevates pathogen resistance and induces pathogenesis-related gene expression in tobacco.

In animals, plants and fungi, cholera toxin (CTX) can activate signalling pathways dependent on heterotrimeric GTP binding proteins (G-proteins). We transformed tobacco plants with a chimeric gene encoding the A1 subunit of CTX regulated by a light-inducible wheat Cab-1 promoter. Tissues of transgenic plants expressing CTX showed greatly reduced susceptibility to the bacterial pathogen Pseudomonas tabaci, accumulated high levels of salicylic acid (SA) and constitutively expressed pathogenesis-related (PR) protein genes encoding PR-1 and the class II isoforms of PR-2 and PR-3. In contrast, the class I isoforms of PR-2 and PR-3 known to be induced in tobacco by stress, by ethylene treatment and as part of the hypersensitive response to infection, were not induced and displayed normal regulation. In good agreement with these results, microinjection experiments demonstrated that CTX or GTP-gamma-S induced the expression of a PR1-GUS reporter gene but not that of a GLB-GUS reporter gene containing the promoter region of a gene encoding the class I isoform of PR-2. Microinjection and grafting experiments strongly suggest that CTX-sensitive G-proteins are important in inducing the expression of a subset of PR genes and that these G-proteins act locally rather than systemically upstream of SA induction.

Intracellular Agrobacterium can transfer DNA to the cell nucleus of the host plant.

Agrobacterium tumefaciens is a Gram-negative, soil-borne bacterium responsible for the crown gall disease of plants. The galls result from genetic transformation of plant cells by the bacteria. Genes located on the transferred DNA (T-DNA), which is part of the large tumor-inducing (Ti) plasmid of Agrobacterium, are integrated into host plant chromosomes and expressed. This transfer requires virulence (vir) genes that map outside the T-DNA on the Ti plasmid and that encode a series of elaborate functions that appear similar to those of interbacterial plasmid transfer. It remains a major challenge to understand how T-DNA moves from Agrobacterium into the plant cell nucleus, in view of the complexity of obstacles presented by the eukaryotic host cell. Specific anchoring of bacteria to the outer surface of the plant cell seems to be an important prelude to the mobilization of the T-DNA/protein complex from the bacterial cell to the plant cell. However, the precise mode of infection is not clear, although a requirement of wounded cells has been documented. By using a microinjection approach, we show here that the process of T-DNA transfer from the bacteria to the eukaryotic nucleus can occur entirely inside the plant cell. Such transfer is absolutely dependent on induction of vir genes and a functional virB operon. Thus, A. tumefaciens can function as an intracellular infectious agent in plants.

In vitro development of globular zygotic wheat embryos.

We have established in vitro culture conditions for globular zygotic wheat embryos (Triticum aestivum L.). Their nutritional requirements have been systematically investigated. The initial sucrose concentration, as well as the sucrose concentration during the culture, a 6-benzylaminopurine supplement, the use of nitrates and ammonium as nitrogen source have a major influence on the embryo development. Proline has an inhibitory effect on the germination. A double layer system with different media was used to give a continuous variation of the medium composition with time. These culture conditions allowed normal direct embryogenesis in up to 47% of the globular embryos.