The response regulator ARR2: a pollen-specific transcription factor involved in the expression of nuclear genes for components of mitochondrial complex I in Arabidopsis.

Two-component signal systems regulate a variety of cellular activities. They involve at least two common signalling molecules: a signal-sensing kinase and a response regulator that mediates the output response. Multistep systems also require proteins containing phosphotransfer domains. Here we report that the response regulator ARR2 from Arabidopsis is predominantly expressed in pollen and is localized in the nuclear compartment of the plant cell. Furthermore, ARR2 is transcriptionally active in yeast and binds to the promoters of nuclear genes for several components of mitochondrial respiratory chain complex I (nCI) from Arabidopsis. The nuclear nCI genes are up-regulated in pollen during spermatogenesis. The transcription factor functions of ARR2 are mediated by its C-terminal output domain. Our data identify ARR2 as the first eukaryotic response regulator which functions as a transcription factor at a known promoter sequence. Yeast two-hybrid analysis and in vitro interaction studies suggest that ARR2 very probably forms part of a multistep two-component signalling mechanism that includes HPt proteins like AHP1 or AHP2. These findings point to an as yet unidentified signal transduction system that may regulate aspects of floral and mitochondrial gene expression.

[Acute precordial pain: 100 cases in 3 years]. / Dolor precordial agudo: 100 casos en 3 años.

OBJECTIVE: To find the effectiveness of diagnoses of acute precordial pain seen as an emergency at our centre. DESIGN: Observational, descriptive and retrospective study. SETTING: Urban primary care centre. PATIENTS: The 100 most recent patients who attended as an emergency with their first episode of acute precordial pain were included. STUDY PERIOD: December 1994 to March 1998. Home visits, patients without medical records and those seen on repeated attendance for precordialgia were excluded. MEASUREMENTS AND MAIN RESULTS: The emergency diagnosis and the diagnosis recorded afterwards in the clinical history of 100 people with acute precordialgia, aged 54.9 (16.7 years; 56% [n = 56] women), were gathered. Ischaemic cardiopathy (41%, n = 41) and mechanical precordialgia (36%, n = 36) were the most common initial diagnoses. We found 66.6% sensitivity and 81.4% specificity in the detection of ischaemic cardiopathy. The proportion of diagnostic errors was not linked to the pathological history of anxiety, ischaemic cardiopathy or oesophageal disease. CONCLUSIONS: 41% of precordialgias are diagnosed as presumably ischaemic and are potentially serious, although only 50% of them are confirmed as such. Our sensitivity in their diagnosis is comparable to that of other studies.

Dolor precordial agudo: 100 casos en 3 años / Acute precordial pain: 100 cases in three years

Objetivo.Conocer la efectividad diagnóstica frente al dolor precordial agudo atendido de urgencias en nuestro centro. Diseño. Estudio observacional, descriptivo y retrospectivo. Emplazamiento.Centro urbano de atención primaria. Pacientes. Se incluyeron los 100 últimos pacientes que acudieron de urgencias con el primer episodio de dolor precordial agudo. Período de estudio: diciembre de 1994 a marzo de 1998. Se excluyeron las visitas domiciliarias, los pacientes sin historia clínica y los atendidos en visitas sucesivas por precordialgia. Mediciones y resultados principales.Se recoge el diagnóstico en urgencias y el registrado a posteriori en la historia clínica de 100 sujetos con precordialgia aguda, de edad 54,9 ñ 16,7 años, 56 de ellos mujeres (56 por ciento). La cardiopatía isquémica y la precordialgia mecánica fueron los diagnósticos iniciales más frecuentes (41 por ciento [n = 41] y 36 por ciento [n = 36], respectivamente). Tenemos una sensibilidad del 66,6 por ciento y una especificidad del 81,4 por ciento para la detección de cardiopatía isquémica. La proporción de errores diagnósticos no se asociaba a los antecedentes patológicos de ansiedad, cardiopatía isquémica o esofágica. Conclusiones.Un 41 por ciento de las precordialgias son de presunto diagnóstico isquémico y potencialmente graves, aunque sólo llegan a confirmarse como tales el 50 por ciento de ellas. Nuestra sensibilidad para su diagnóstico es comparable a la de otros estudios (AU)

Transcription signals of mitochondrial and nuclear genes for mitochondrial proteins in dicot plants.

Mitochondria contain several large multisubunit enzyme complexes that are composed of proteins encoded in the nuclear and mitochondrial genomes. Particularly for correct assembly of these enzyme complexes, expression of the respective mitochondrial and nuclear genes has to be coordinated to ensure correct stoichiometries of the protein subunits. Part of this control and the response to specific demands is exercised at the level of transcription. To determine the respective transcription signals we have analyzed the mitochondrial promoters in dicot plants and the promoter structure for nuclear-encoded genes of the respiratory chain complex I. We summarize the results of these investigations and extend the mitochondrial promoter survey to the mitochondrial genome in Arabidopsis thaliana.

Promoters of nuclear-encoded respiratory chain complex I genes from Arabidopsis thaliana contain a region essential for anther/pollen-specific expression.

Regulatory promoter regions responsible for the enhanced expression in anthers and pollen are defined in detail for three nuclear encoded mitochondrial Complex I (nCl) genes from Arabidopsis thaliana. Specific regulatory elements were found conserved in the 5' upstream regions between three different genes encoding the 22 kDa (PSST), 55 kDa NADH binding (55 kDa) and 28 kDa (TYKY) subunits, respectively. Northern blot analysis and transgenic Arabidopsis plants carrying progressive deletions of the promoters fused to the beta-glucuronidase (GUS) reporter gene by histochemical and fluorimetric methods showed that all three promoters drive enhanced expression of GUS specifically in anther tissues and in pollen grains. In at least two of these promoters the -200/-100 regions actively convey the pollen/anther-specific expression in gain of function experiments using CaMV 35S as a minimal promoter. These nCl promoters thus contain a specific regulatory region responding to the physiological demands on mitochondrial function during pollen maturation. Pollen-specific motifs located in these regions appear to consist of as little as seven nucleotides in the respective promoter context.

Physiological, biochemical and molecular aspects of mitochondrial complex I in plants

Respiratory complex I of plant mitochondria has to date been investigated with respect to physiological function, biochemical properties and molecular structure. In the respiratory chain complex I is the major entry gate for low potential electrons from matrix NADH, reducing ubiquinone and utilizing the released energy to pump protons across the inner membrane. Plant complex I is active against a background of several other NAD(P)H dehydrogenases, which do not contribute in proton pumping, but permit and establish several different routes of shuttling electrons from NAD(P)H to ubiquinone. Identification of the corresponding molecular structures, that is the proteins and genes of the different NADH dehydrogenases, will allow more detailed studies of this interactive regulatory network in plant mitochondria. Present knowledge of the structure of complex I and the respective mitochondrial and nuclear genes encoding various subunits of this complex in plants is summarized here. Copyright 1998 Elsevier Science B.V.

Impairment of tapetum and mitochondria in engineered male-sterile tobacco plants.

Flowers of tobacco transformed with an unedited copy of the mitochondrial atp9 gene sequence fused to the yeast coxIV mitochondrial targeting presequence, showed several anther abnormalities leading to pollen abortion. The gene was expressed in vegetative and reproductive tissues of the plant. Cytological analysis revealed that tapetum development was impaired. Mitochondria of the tapetum cells were severely affected showing characteristic signs of degeneration: loss of cristae and swelling. These mitochondrial modifications were correlated with the presence of the transcript and translated product of the 'unedited' atp9 and a significant decrease in oxygen consumption in non-photosynthetic tissues. The main effect of the unedited atp9 expression in transgenic plants was male sterility.

Transgenic male-sterile plant induced by an unedited atp9 gene is restored to fertility by inhibiting its expression with antisense RNA.

We have previously shown that the expression of an unedited atp9 chimeric gene correlated with male-sterile phenotype in transgenic tobacco plant. To study the relationship between the expression of chimeric gene and the male-sterile trait, hemizygous and homozygous transgenic tobacco lines expressing the antisense atp9 RNA were constructed. The antisense producing plants were crossed with a homozygous male-sterile line, and the F1 progeny was analyzed. The offspring from crosses between homozygous lines produced only male-fertile plants, suggesting that the expression antisense atp9 RNA abolishes the effect of the unedited chimeric gene. In fact, the plants restored to male fertility showed a dramatic reduction of the unedited atp9 transcript levels, resulting in normal flower development and seed production. These results support our previous observation that the expression of unedited atp9 gene can induce male sterility.

Expression of one of the members of the Arabidopsis chaperonin 60 beta gene family is developmentally regulated and wound-repressible.

To study the pattern of gene regulation of the plastid chaperonin 60 beta gene family a chimaeric gene was constructed fusing the 5'-flanking region of the chaperonin 60 beta B3 gene to the beta-glucuronidase reporter gene. Histochemical and fluorometric analysis of the GUS activity present in transgenic plants harbouring this gene construct showed that the B3 promoter is expressed in leaves, stem, petioles and several flower tissues. The pattern of cell type-specific expression in stems and flowers was found to be developmentally regulated. Expression of the B3 promoter was found not to be heat-inducible, but highly repressed by wounding. The rapid decay in GUS activity upon wounding indicates that, at least under some physiological conditions, the gene product of this reporter gene is not as stable as has been previously thought.

Isolation and characterization of genes encoding chaperonin 60 beta from Arabidopsis thaliana.

Chaperonins (Cpn) are implicated in the folding and assembly of multimeric proteins in plastids and mitochondria of eukaryotes and in prokaryotes. Plastid Cpn is composed of two different polypeptides termed Cpn60 alpha and Cpn60 beta. We have isolated cDNA and genomic clones encoding Cpn60 beta from Arabidopsis thaliana. The steady-state level of the cpn60 beta mRNAs is higher in etiolated leaves and sucrose-treated plants as compared to control leaves. The A. thaliana cpn60 beta gene family consists of at least three different coding units. It was confirmed that Cpn beta-encoding genes have a high level of conservation among plants.

Sucrose Synthase Expression during Cold Acclimation in Wheat.

When wheat (Triticum aestivum) seedlings are exposed to a cold temperature (2-4 degrees C) above 0 degrees C, sucrose accumulates and sucrose synthase activity increases. The effect of a cold period on the level of sucrose synthase (SS) was investigated. Using antibodies against wheat germ SS, Western blots studies showed that the amount of the SS peptide increased during 14 days in the cold, when plants were moved from 23 degrees C to 4 degrees C. The level of SS diminished when plants were moved back to 23 degrees C. Northern blots of poly(A)(+) RNA, confirmed a five- to sixfold induction of SS in wheat leaves during cold acclimation. These results indicate that SS is involved in the plant response to a chilling stress.

Ectopic expression of the Y (Ley) antigen defined by monoclonal antibody 12-4LE in distal colonic adenocarcinomas.

Monoclonal antibody (MAb) 12-4LE reacts specifically with the alpha Fuc(1-2) beta Gal(1-4) [alpha Fuc(1-3)]GlcNAc-R synthetic oligosaccharide and consequently characterizes the Y (Ley) antigen. In normal individuals, this MAb reacts more strongly on samples from blood group O persons, indicating that the Y structure is better recognized when terminal A or B sugars are not added to the Y structure. In fetal and normal adult gastrointestinal tract, this antibody reacts with the epithelium of stomach, small intestine and proximal colon, but not of distal colon. In the adult, cells from the surface epithelium of the gastric, small intestinal and cecal mucosae express the Y antigen according to the secretor phenotype of each individual, thus characterizing the so-called "upward differentiation" pattern. In contrast, mucus cells of the pylorus and duodenal Brünner glands, as well as Paneth cells, always express the Y antigen irrespective of secretor phenotype, thereby characterizing the so-called "downward differentiation" pattern. Proximal fetal colonic mucosa has the same genetic control as the downward differentiation pattern of the adult. Distal fetal colonic mucosa is negative with anti-Y, as in the adult. Y antigen was not expressed in hyperplastic (10 cases), juvenile (5 cases) or adenomatous (43 cases) polyps, except for some spreading villous adenomas in which rare Y-positive foci could be observed but which were not specifically associated with dysplastic glands. Polyps from familiar polyposis did not express this antigen. In adenocarcinomas, the Y antigen was expressed in 41/45 (91%) of distal tumors and 15/35 (43%) of cecal tumors, independently of ABO phenotype. The ectopic expression of this Y antigen on distal colon adenocarcinomas may be a useful tool in the detection of distal colonic carcinomas.

Distribution of GICA in normal gastrointestinal and endocervical mucosae and in mucinous ovarian cysts using antibody NS 19-9.

Through the immunoperoxidase method using antibody 19-9, we demonstrated the presence of gastrointestinal cancer-associated antigen (GICA) in normal gastrointestinal and endocervical mucosae and in benign mucinous ovarian cysts of pure endocervical type in Le(a+b-) and Le(a-b+) patients exclusively. However, GICA was more quantitatively expressed in Le(a+b-) than in Le(a-b+) patients. GICA was always encountered in the ducts of esophageal glands, Wirsung's ducts, goblet cells of villosities near the ileo-colonic junction, and the mucus columnar cells of the endocervical mucosae or ovarian mucinous tumor epithelium. In other areas of the gastrointestinal tract, GICA was sometimes found in the mucus cells of esophageal glands, near the neck cells of gastric mucosae, in goblet cells of the upper part of the small intestinal villosities, and in colonic Lieberkühn glands, especially in the cell maturation compartment. GICA therefore appears to be a normal component of gastrointestinal and endocervical mucosae.

Biosynthesis of Lewis antigenic glycolipid by cell-free extracts of human intestinal tumor cells cultured in serum-free medium.

Extracts of the human intestinal tumor cell line SW1116 were able to stimulate the incorporation of (14C) fucose from GDP-(14C) fucose into organically soluble glycolipid. The reaction required a purified glycolipid preparation from human meconium as lipid acceptor. The active glycolipid co-migrated with standard globoside on high performance thin-layer chromatography (HPTLC) and had molecular species (M + H) under fast-atom bombardment mass spectrometry of 1199, 1245 and 1269. Globoside itself was inactive and asialo GM1b had low activity. The radioactive products co-purified with Lewis a and Lewis b and co-migrated principally (60-90%) with Lewis b monoclonal antibody binding cellular glycolipids on HPTLC. Analysis of fucosidase digests suggested the presence of two different fucosyl-hexose linkages one of which was susceptible to cleavage. We conclude that the data are consistent with fucosylation of lactotetraosyl ceramide to Lewis a and Lewis b antigenic glycolipids.

Alterations in expression and synthesis of glycolipid antigens in human colonic tumor cell lines.

Incubation of human intestinal SW1116 tumor cells in serum-free medium containing butyric acid reduced their capacity to synthesize gastrointestinal carcinoma-associated (GICA) glycolipid antigen 4- to 8-fold as determined by a radioimmunobinding assay using anti-GICA monoclonal antibody:high-performance thin-layer chromatography; autoradiography; and densitometry. The structure of GICA has been described as a sialylated Lea glycolipid (J. L. Magnani, B. Nilsson, M. Brockhaus, D. Zopf, Z. Steplewski, A. Koprowski, and V. Ginsburg. J. Biol. Chem., 257: 14365-14369, 1982). Tritiated fucose incorporation into GICA was reduced per cell (7-fold), per mg protein (5-fold), and per mg lipid (4-fold). A purified organically soluble glycolipid fraction from control SW1116 cells contained more Lewis antigen than did butyrate-treated cells as determined by the high-performance thin-layer chromatography radioimmunobinding assay. Incorporation of radioactivity from [3H]fucose and guanosine diphosphate [14C]fucose into Lewis antigens in butyrate-treated cells was 2- to 3-fold lower than in control cells. HT29 cells carry the blood type of the original donor, Blood Group A. Isotope incorporation into A glycolipid antigen was reduced 2- to 8-fold upon exposure to butyrate. Commensurate with these results was a dramatic reduction in cell population-doubling rate. We propose that synthesis of these fucolipid antigens is associated more with dividing, undifferentiated tumor cell populations. The diminution in antigen levels may derive from diminished cells' capacity for fucosylation in the presence of butyrate.