LexA-binding sequences in Gram-positive and cyanobacteria are closely related.

The lexA gene of the cyanobacterium Anabaena sp. strain PCC7120 has been cloned by PCR amplification with primers designed after TBLASTN analysis of its genome sequence using the Escherichia coli LexA sequence as a probe. After over-expression in E. coli and subsequent purification, footprinting experiments demonstrated that the Anabaena LexA protein binds to the sequence TAGTACTAATGTTCTA, which is found upstream of its own coding gene. Directed mutagenesis and sequence comparison of promoters of other Anabaena genes, as well as those of several cyanobacteria, allowed us to define the motif RGTACNNNDGTWCB as the LexA box in this bacterial phylum. Substitution of a single nucleotide in this motif present in the Anabena lexA promoter is sufficient to enable it to bind the Bacillus subtilis LexA protein. These data indicate that Cyanobacteria and Gram-positive bacteria are phylogenetically closely related.

Ferredoxin-dependent iron-sulfur flavoprotein glutamate synthase (GlsF) from the Cyanobacterium synechocystis sp. PCC 6803: expression and assembly in Escherichia coli.

The unicellular cyanobacterium Synechocystis sp. PCC 6803 contains two different glutamate synthases whose genes, gltB and glsF (previously known as gltS), have been cloned (F. Navarro et al., 1995, Plant Mol. Biol. 27, 753-767). The glsF gene has been expressed in the glutamate auxotrophic Escherichia coli strain CLR207 RecA, but the corresponding protein does not complement the auxotrophy. The transformed strain showed ferredoxin-dependent glutamate synthase (Fd-GOGAT) activity, demonstrating the capability of E. coli for providing and correctly assembling both the iron-sulfur center and the flavin cofactor of the enzyme. Fd-GOGAT (GlsF) is correctly cleaved at Cys37 to form the mature enzyme in E. coli, as occurs with the large subunit of its own NADPH-GOGAT. The recombinant Fd-GOGAT has been purified to electrophoretic homogeneity, using as the main purification step a ferredoxin-affinity chromatography. The pure enzyme, with a molecular mass of about 180 kDa, shows an absorption spectrum characteristic of iron-sulfur flavoproteins. The analyses of the prosthetic groups indicate that Fd-GOGAT contains only one FMN, but no FAD, and one [3Fe-4S](+,0) cluster per molecule. Oxidation-reduction titration, using absorbance changes of the FMN group in the visible region, gave a midpoint redox potential of -200 +/- 25 mV at pH 7.5. The recombinant enzyme is strictly ferredoxin-dependent and shows apparent K(M) values similar to those of the native Synechocystis protein: 4.5 vs 3.5 microM, 2.2 vs 2.5 mM, and 0.6 vs 0.5 mM for ferredoxin, glutamine, and 2-oxoglutarate, respectively. The addition of the reductant dithionite to the enzyme resulted in the loss of the absorption peak at 436 nm, characteristic of oxidized flavins, which was restored by the anaerobic addition of 2-oxoglutarate, in the presence of glutamine.

The presence of glutamate dehydrogenase is a selective advantage for the Cyanobacterium synechocystis sp. strain PCC 6803 under nonexponential growth conditions.

The unicellular cyanobacterium Synechocystis sp. strain PCC 6803 has two putative pathways for ammonium assimilation: the glutamine synthetase-glutamate synthase cycle, which is the main one and is finely regulated by the nitrogen source; and a high NADP-dependent glutamate dehydrogenase activity (NADP-GDH) whose contribution to glutamate synthesis is uncertain. To investigate the role of the latter, we used two engineered mutants, one lacking and another overproducing NADP-GDH. No major disturbances in the regulation of nitrogen-assimilating enzymes or in amino acids pools were detected in the null mutant, but phycobiline content, a sensitive indicator of the nutritional state of cyanobacterial cells, was significantly reduced, indicating that NADP-GDH plays an auxiliary role in ammonium assimilation. This effect was already prominent in the initial phase of growth, although differences in growth rate between the wild type and the mutants were observed at this stage only at low light intensities. However, the null mutant was unable to sustain growth at the late stage of the culture at the point when the wild type showed the maximum NADP-GDH activity, and died faster in ammonium-containing medium. Overexpression of NADP-GDH improved culture proliferation under moderate ammonium concentrations. Competition experiments between the wild type and the null mutant confirmed that the presence of NADP-GDH confers a selective advantage to Synechocystis sp. strain PCC 6803 in late stages of growth.

Daily variation patterns of airborne allergenic pollen in southwestern Spain.

The study was carried out using a Burkard sampler installed on the roof terrace of the School of Pharmacy, Seville, for two years (1995 and 1996). Eight pollen types described in the literature as having allergenic activity were chosen. They were Poaceae, Olea europaea, Chenopodiaceae/Amaranthaceae, Plantago, Rumex, Urticaceae (including Parietaria), Cupressaceae, and Platanus hispanica. The types were grouped according to the similarity of their pattern of intradiurnal variation in pollen concentration. The following associations were established by multivariate analysis: Urticaceae and Chenopodiaceae/Amaranthaceae (appearing mainly between 11:00 and 20:00), Olea europaea and Plantago (12:00 to 19:00), Poaceae and Rumex (appearing throughout the day), and Cupressaceae and Platanus hispanica (8:00 to 14:00). The patterns of intradiurnal variation were similar both years for each type, despite the fact that the two years were climatologically different (1995 was dry and 1996 wet). We conclude that these behavior patterns are endogenous to the plants, and are hardly affected by meteorological parameters.

Airborne grass (Poaceae) pollen in southern Spain. Results of a 10-year study (1987-96).

This work reports an exhaustive study of the aerobiology of the Gramineae in Seville, Spain, which is typical of coastal Mediterranean areas. Sampling was done with a Cour trap installed on the roof terrace of the School of Pharmacy, Seville, from 1987 to 1996, both inclusive. The climatic pattern of that period was characterized by two exceptionally wet years (1989 and 1996), between which were 5 consecutive years of drought (1990-5). This typically Mediterranean climate affects grass aerobiology. The annual amounts of total grass pollen are low, never exceeding 2500 grains/m3. The start, length, and intensity of the pollen season are significantly correlated with preseasonal meteorologic factors (precipitation and temperature), but intraseasonal meteorologic conditions have no effect on the three variables. The relationships are stated by three equations that, while further years of observations are anticipated, can be considered models to forecast the characteristics of the pollen season: the starting date depends on the mean temperatures of January and February, and the length and intensity of the season depend on the rainfall between the beginning of January and the starting date of the season. For the study period, the weekly concentrations (pollen curves) throughout the year showed no typical pattern of variation over the years, so that it was impossible to make mid- and long-term forecasts of the variation in weekly concentration. The most noteworthy aspects of grass pollen curves are a long pollen season, which starts in February or March and lasts until September or October; peaks of higher concentration (> 100 grains/m3) in May and June, associated with increases in temperature and absence of precipitation; and other peaks in the summer months that may be as high as the spring peaks.

Aerobiological study of Chenopodiaceae and Amaranthaceae in the Mediterranean area of southwestern Spain.

The study area is characterized by a maritime Mediterranean climate, abundant presence of Chenopodiaceae in the vegetation, and a high incidence of pollinosis caused by the pollen of this family. A Hirst-type sampler was used to determine pollen concentrations in the air of the city of Huelva during 3 consecutive years (1995-1997). The total annual amount of Chenopodiaceae-Amaranthaceae pollen was between 10.59% and 6.28% of the pollen spectrum of the city, depending on the year. The annual pattern of variation in pollen concentration (5-day means) was very similar in the 3 years, and no statistically significant differences were found between years. This pattern is characterized by concentrations not exceeding 20 grains/m3 between April and the beginning of August, with an obvious seasonal variation in the second half of August and September, when the 5-day mean concentrations exceeded 40 grains/m3 and the daily maxima exceeded 100 grains/m3. This coincided with the flowering of most of the species in the group. The meteorological parameters with a statistically significant effect on daily pollen concentration during the pollen season (August 15 to September 20) included mean temperature and south wind (positive correlations) and relative humidity of the air (negative correlation). The highest intradiurnal concentrations were found between 10:00 a.m. and 5:00 p.m.

Olea europaea airborne pollen in southern Spain.

BACKGROUND: Olea europaea pollen is one of the most abundant constituent pollens in Seville (southern Spain). It is responsible for many documented cases of pollinosis in the area. OBJECTIVE: To contribute to the useful knowledge of Olea europaea for allergists. METHODS: The number of Olea europaea pollen grains in the atmosphere was recorded during 8 consecutive years (1987-1994), using a Cour collector. RESULTS: The concentration of Olea europaea pollen was as high as 250 to 1015 grains/m3 only during 1 or 2 weeks in April and May. Overall annual production of O. europaea pollen alternated between years. The beginning of the main pollination period was related to the mean temperature of the preceding months (February and March). Pollination occurred when the mean temperature in both months was higher than 14 degrees C; conversely it was delayed when the mean temperature was lower. Main pollination period length depended upon both temperature and rainfall during this period: temperatures higher than 19.5 degrees C and absence of rainfall shortened the main pollination period, while lower temperatures (15 to 18 degrees C) together with rainfall rates above 100 mm lengthened it. CONCLUSIONS: Climatic variables such as preceding mean temperature and rainfall impact on pollen anthesis of Olea europaea affect onset and duration of pollination. A consideration of yearly cycles of pollen production as well as these variables should allow pollen forecasting.

The NADP-glutamate dehydrogenase of the cyanobacterium Synechocystis 6803: cloning, transcriptional analysis and disruption of the gdhA gene.

The gdhA gene of Synechocystis PCC 6803, which encodes an NADP-dependent glutamate dehydrogenase (NADP-GDH), has been cloned by complementation of an Escherichia coli glutamate auxotroph. This gene was found to code for a polypeptide of 428 amino acid residues, whose sequence shows high identity with those of archaebacteria (42-47%), some Gram-positive bacteria (40-44%) and mammals (37%). The minimal fragment of Synechocystis DNA required for complementation (2kb) carries the gdhA gene preceded by an open reading frame (ORF2) encoding a polypeptide of 130 amino acids. ORF2 and gdhA are co-transcribed as a 1.9 kb mRNA, but shorter transcripts including only gdhA were also detected. Two promoter regions were identified upon transcriptional fusion to the cat reporter gene of a promoter probe plasmid. Transcription from the promoter upstream of ORF2 was found to be regulated depending on the growth phase of Synechocystis, in parallel to NADP-GDH activity. This promoter is expressed in Escherichia coli too, in contrast to the second promoter, located between ORF2 and gdhA, which was silent in E. coli and did not respond to the stage of growth in Synechocystis. Disruption of the cyanobacterial gdhA gene with a chloramphenicol resistance cassette yielded a mutant strain totally lacking NADP-GDH activity, demonstrating that this gene is not essential to Synechocystis 6803 under our laboratory conditions.

Existence of two ferredoxin-glutamate synthases in the cyanobacterium Synechocystis sp. PCC 6803. Isolation and insertional inactivation of gltB and gltS genes.

The first two genes of ferredoxin-dependent glutamate synthase (Fd-GOGAT) from a prokaryotic organism, the cyanobacterium Synechocystis sp. PCC 6803, were cloned in Escherichia coli. Partial sequencing of the cloned genomic DNA, of the 6.3 kb Hind III and 9.3 kb Cla I fragments, confirmed the existence of two different genes coding for glutamate synthases, named gltB and gltS. The gltB gene was completely sequenced and encodes for a polypeptide of 1550 amino acid residues (M(r) 168,964). Comparative analysis of the gltB deduced amino acid sequence against other glutamate synthases shows a higher identity with the alfalfa NADH-GOGAT (55.2%) than with the corresponding Fd-GOGAT from the higher plants maize and spinach (about 43%), the red alga Antithamnion sp. (42%) or with the NADPH-GOGAT of bacterial source, such as Escherichia coli (41%) and Azospirillum brasilense (45%). The detailed analysis of Synechocystis gltB deduced amino acid sequence shows strongly conserved regions that have been assigned to the 3Fe-4S cluster (CX5CHX3C), the FMN-binding domain and the glutamine-amide transferase domain. Insertional inactivation of gltB and gltS genes revealed that both genes code for ferredoxin-dependent glutamate synthases which were nonessential for Synechocystis growth, as shown by the ferredoxin-dependent glutamate synthase activity and western-blot analysis of the mutant strains.

Latitudinal study of allergenic pollen in two Spanish cities.

A comparative study was performed on the pollen spectra in 1990 in two Spanish cities with different vegetation and climate, one situated on the south coast (Huelva) and the other in the north and inland (Palencia). The flowering of Alnus and Fraxinus occurred earlier in the north, while that of grasses (Plantago, Poaceae and Urticaceae) occurred earlier and lasted longer in the south, with milder temperature conditions resulting in no fall in pollen content between winter and spring. The absence of olive pollen in Palencia reaffirms that the pollen spectrum of a locality is a consequence of the surrounding vegetation. The geographical situation of Palencia, in the interior of Spain, results in higher percentages of Poaceae and Quercus. The enormous pollution of Huelva favors the greater development of the Urticaceae family and the greater presence of its pollen in the air.

Effect of Glucose Utilization on Nitrite Excretion by the Unicellular Cyanobacterium Synechocystis sp. Strain PCC 6803.

Up to 1 mM nitrite was excreted by Synechocystis strain 6803 cells growing under mixotrophic or photoheterotrophic conditions. This excretion is not due to a lower ratio of nitrite and nitrate reductase activities in the presence of glucose but seems to be related to a shortage of reduced ferredoxin, their electron donor, as a result of a decrease in noncyclic photosynthetic flow observed under these circumstances. Because about 60% of the reduced nitrate is excreted, the potential utilization of cyanobacteria for removal of nitrate from contaminated waters containing high concentrations of organic compounds is questioned.

Pollen calendar of Seville and its relation to allergies.

We have carried out a study on the annual and weekly variation of airborne pollen in the air of Seville (Andalusia, Southern Spain) during 3 consecutive years using two Cour's collectors. We provide the pollen calendar of this city, which shows the annual distribution of the most important pollen types. Our results led us to check the validity of the pollen extracts usually used at the hospitals of the city to diagnose pollinosis.

A study of the aeromycoflora of Cádiz: relationship to anthropogenic activity.

We describe a quantitative and qualitative study of the fungal spores found in the air of Cádiz during 1989 using a Cour-type trap. The results of this study can be extrapolated to other coastal cities of southern Europe with a Mediterranean climate. The spores identified have been classified into 25 taxonomic categories. The most abundant were Cladosporium, Chaetomium and Ustilago, and the most frequent, in addition to those mentioned, were Alternaria, Ascophyta and Venturia. The great abundance of Cladosporium is in accordance with the coastal situation of the city. Cladosporium, Alternaria, Curvularia and Stemphylium reached maximum concentrations jointly in October, 1989. They showed mutual cross-reactions. Ustilago and Nigrospora appeared during the period of cereal harvesting and storage.

Purification and characterization of the ferredoxin-glutamate synthase from the unicellular cyanobacterium Synechococcus sp. PCC 6301.

Ferredoxin-glutamate synthase from the unicellular cyanobacterium Synechococcus sp. PCC 6301 has been purified using, as main steps, ethanol fractionation in the presence of high ionic strength, ion-exchange chromatography and ferredoxin-Sepharose affinity chromatography. The overall process yielded an homogeneous enzyme with a specific activity of 30 U/mg protein, after a purification of 2800-fold with a recovery of 43%. The molecular mass of the native protein was 156 kDa, as calculated from its Stokes radius (rS, 4.32 nm) and sedimentation coefficient (S20,w, 8.46 S). The size was also estimated by SDS/PAGE as 160 kDa, indicating that the native protein was a monomer. The enzyme exhibited absorption maxima at 279, 370 and 438 nm and a A279/A438 absorbance ratio of 11. One molecule of FMN, but not FAD, was found/molecule native protein. The addition of dithionite resulted in the loss of the absorption peak at 438 nm, which was restored by the addition of 2-oxoglutarate, thus indicating that the prosthetic group is functional in catalysis. Classical hyperbolic kinetics with substrate inhibition was seen for 2-oxoglutarate. The Km values determined for glutamine and ferredoxin were 0.7 mM and 7 microM, respectively, and the apparent Km for 2-oxoglutarate was estimated to be 1.7 mM. Azaserine and 6-diazo-5-oxo-L-norleucine were potent inhibitors of the activity, while pyridoxal 5-phosphate, known to react with Lys residues, partially inactivated the enzyme. This ferredoxin-dependent glutamate synthase is, as far as we know, the first purified from prokaryotic organisms and resembles its counterpart from chloroplasts, suggesting that cyanobacterial glutamate synthase may have been the ancestor of ferredoxin-glutamate synthase in plants.

[Exposure of the facial recess through the ear canal. Value in posterosuperior retraction pockets (initial results)]. / Mise à plat du recessus facial par voie du conduit. Intérêt dans les poches de rétraction tympaniques postéro-supérieures (résultats préliminaires).

19 patients had surgery for progressive [17] and/or symptomatic [2] posterior attic retraction pockets involving the facial recess. Exposition of the suprapyramidal region was obtained after endaural incision by thinning the posterior wall of the ear canal and removing the posterior-superior portion of the tympanic sulcus. This technique is less complicated than intact canal wall tympanoplasty with mastoidectomy. Yet gives similar functional results. After a mean follow-up of 20.3 months, we have observed no residual cholesteatoma and no recurrent retraction pockets. Unlike posterior tympanoplasty, this technique makes it possible to meticulously remove the osteitic bone invariably found in the facial recess when there is infection of the retraction pocket.

Light-mediated regulation of glutamine synthetase activity in the unicellular cyanobacterium Synechococcus sp. PCC 6301.

Glutamine synthetase (GS; EC 6.3.1.2) activity from the unicellular cyanobacterium Synechococcus sp. strain PCC 6301 shows a short-term regulation by light-dark transitions. The enzyme activity declines down to 30% of the original level after 2 h of dark incubation, and can be fully reactivated within 15 min of re-illumination. The loss of activity is not due to protein degradation, but rather to a reversible change of the enzyme, as deduced from the GS-protein levels determined in dark-incubated cells using polyclonal antibodies raised against Synechococcus GS. Incubation with 3-(3-4-dichlorophenyl)-1,1-dimethylurea (DCMU) also provokes GS inactivation, indicating that an active electron flow between both photosystems is necessary to maintain GS in an active state. On the other hand, the light-mediated reactivation of GS in dark-incubated cells treated with dicyclohexyl-carbodiimide (DCCD) or carbonyl cyanide m-chlorophenylhydrazone (CCCP) indicates that neither changes in the ATP synthesis nor the lack of an electrochemical proton gradient across the thylakoid membrane are directly involved in the regulation process. The inactive form of GS is extremely labile in vitro after disruption of the cells, and is not reactivated by treatment with dithiothreitol or spinach thioredoxin m. These results, taken together with the fact that dark-promoted GS inactivation is dependent on the growth phase, seem to indicate that GS activity is not regulated by a typical redox process and that some other metabolic signal(s), probably related to the ammonium-assimilation pathway, might be involved in the regulation process. In this regard, our results indicate that glutamine is not a regulatory metabolite of Synechococcus glutamine synthetase.

In vitro reactivation of in vivo ammonium-inactivated glutamine synthetase from Synechocystis sp. PCC 6803.

Glutamine synthetase from Synechocystis sp. strain PCC 6803 is inactivated by ammonium addition to cells growing with nitrate as the nitrogen source. The enzyme can be reactivated in vitro by different methods such as alkaline phosphatase treatment, but not phosphodiesterase, by raising the pH of the crude extract to values higher than 8, by increasing the ionic strength of the cell-free extract, or by preincubation with organic solvents, such as 2-propanol and ethanol. These results suggest that the loss of glutamine synthetase activity promoted by ammonium involves the non-covalent binding of a phosphorylated compound to the enzyme and support previous results that rule out the existence of an adenylylation/deadenylylation system functioning in the regulation of cyanobacterial glutamine synthetase.

An NAD-specific glutamate dehydrogenase from cyanobacteria. Identification and properties.

The unicellular cyanobacterium Synechocystis sp. PCC 6803 presents a hexameric NAD-specific glutamate dehydrogenase with a molecular mass of 295 kDa. The enzyme differs from the NADP-glutamate dehydrogenase found in the same strain and is coded by a different gene. NAD-glutamate dehydrogenase shows a high coenzyme specificity, catalyzes preferentially glutamate formation and presents Km values for ammonium, NADH and 2-oxoglutarate of 4.5 mM, 50 microM and 1.8 mM respectively. An animating role for the enzyme is discussed.

Regulation of glutamine synthetase activity in the unicellular cyanobacterium Synechocystis sp. strain PCC 6803 by the nitrogen source: effect of ammonium.

Glutamine synthetase activity from Synechocystis sp. strain PCC 6803 is regulated as a function of the nitrogen source available in the medium. Addition of 0.25 mM NH4Cl to nitrate-grown cells promotes a clear short-term inactivation of glutamine synthetase, whose enzyme activity decreases to 5 to 10% of the initial value in 25 min. The intracellular levels of glutamine, determined under various conditions, taken together with the results obtained with azaserine (an inhibitor of transamidases), rule out the possibility that glutamine per se is responsible for glutamine synthetase inactivation. Nitrogen starvation attenuates the ammonium-mediated glutamine synthetase inactivation, indicating that glutamine synthetase regulation is modulated through the internal balance between carbon-nitrogen compounds and carbon compounds. The parallelism observed between the glutamine synthetase activity and the internal concentration of alpha-ketoglutarate suggests that this metabolite could play a role as a positive effector of glutamine synthetase activity in Synechocystis sp. Despite the similarities of this physiological system to that described for enterobacteria, the lack of in vivo 32P labeling of glutamine synthetase during the inactivation process excludes the existence of an adenylylation-deadenylylation system in this cyanobacterium.

[Role of emergency subtotal colectomy in neoplastic obstructions of the left colon]. / Place de la colectomie subtotale en urgence dans les occlusions néoplasiques du côlon gauche.

From 1984 to 1990, 60 patients underwent emergent surgery for a neoplastic obstruction of the left colon. We performed 19 colostomies without initial exeresis and 41 immediate tumoral resections. In the latter group, five subtotal colectomies (S.T.C.) were performed, including four with immediate mechanical anastomosis. Two patients had synchronous cancers and three had pre-perforating cecal lesions. Three patients had an associated general peritonitis. Three of the patients treated with STC died. These were these patients with general peritonitis, two of whom also had hepatic metastases. The data found in the literature on neoplastic obstructions of the left colon treated with STC with immediate anastomosis (227 cases are published) show an overall mortality rate of 8.4% with 24% morbidity, a complication of the anastomosis occurring in 4.5% of all cases.