Operon fusions of Mu d1(Ap,lac) to thel-proline biosynthetic genes ofescherichia coli K-12

Operon fusions to the promoter of either theproA,proB, orproC genes of the proline biosynthetic pathway were obtained by the use of the Mu d1(Ap,lac) bacteriophage. These fusions were further stabilized by transformation with plasmid pGW600 containing the wildtype Mu repressor gene or by transduction with phage pSG1. The level of -galactosidase in the fusion strains was not affected by the presence of exogenously addedl-proline or high concentrations of NaCl in the growth medium. A Tn5 insertion nearproBA increased -galactosidase expression 140- to 200-fold in strains carrying theproA-lac andproB-lac fusions, but the level of this enzyme was unaltered in strains carrying theproC-lac fusion. The Tn5 insertion increased intracellular proline concentrations 8- to 10-fold, suggesting that mechanisms other than allosteric inhibition may regulate proline biosynthesis, but did not confer osmotolerance to cells growing in a medium with a high concentration of salt.     

Dopamine-1-mediated stimulation of phospholipase C activity in rat renal cortical membranes

Phospholipase C (PL-C) mediates transduction of neurotransmitter signals across membranes via hydrolysis of phosphatidylinositol-4,5-bisphosphate (PIP2), leading to generation of second messengers inositol-1,4,5-trisphosphate and diacylglycerol. In this study, dopamine-1 (DA-1) but not dopamine-2 (DA-2) agonists were shown to stimulate PL-C activity in renal cortical membranes. The DA-1 agonist, SKF 82526, stimulated the release of inositol phosphates from renal cortical membranes prelabeled with [3H]myoinositol. The majority of the label (75%) was found in phosphatidylinositol followed by PIP2 (15%) and phosphatidylinositol-4-phosphate (10%). A DA-1 specific effect on PL-C activity was also observed in an in vitro assay of PL-C activity in renal cortical membranes and basolateral and brush border membranes using [3H]PIP2 as the substrate. Dopamine and SKF 82526 stimulated the release of inositol phosphates from added [3H]PIP2 in a concentration-dependent manner. This release was blocked by the DA-1 antagonist SCH 23390 but not by the alpha-adrenergic antagonists phentolamine and prazosin. In contrast, the DA-2 agonist LY 171555 had no effect on inositol phosphate release. Guanosine 5'-(3-O-thio)triphosphate enhanced while guanyl-5'-yl thiophosphate attenuated the DA-1 agonist-stimulated PL-C activity. PL-C activity as measured by [3H]PIP2 hydrolysis had a pH optimum of 6.5, was inhibited by Mg2+ concentrations above 1 mM, was linear with time and protein concentration, and was sensitive to phosphatidylserine and calcium concentrations. We conclude that PL-C is activated by DA-1 but not DA-2 agonists in renal cortical membranes as well as both the basolateral and brush border renal tubular membranes. It is speculated that this action may mediate the natriuretic effects of dopamine in renal tubular epithelia.     

Modulation of microvascular growth and morphogenesis by reconstituted basement membrane gel in three-dimensional cultures of rat aorta: A comparative study of angiogenesis in Matrigel,collagen, fibrin,and plasma clot

Summary Rings of rat aorta cultured in Matrigel, a reconstituted gel composed of basement membrane molecules, gave rise to three-dimensional networks composed of solid cellular cords and occasional microvessels with slitlike lumina. Immunohistochemical and ultrastructural studies showed that the solid cords were composed of endothelial sprouts surrounded by nonendothelial mesenchymal cells. The angiogenic response of the aortic rings in Matrigel was compared to that obtained in interstitial collagen, fibrin, or plasma clot. Morphometric analysis demonstrated that the mean luminal area of the microvascular sprouts and channels was significantly smaller in Matrigel than in collagen, fibrin, or plasma clot. The percentage of patent microvessels in Matrigel was also markedly reduced. Autoradiographic studies of³H-thymidine-labeled cultures showed reduced DNA synthesis by developing microvessels in Matrigel. The overall number of solid endothelial cords and microvessels was lower in Matrigel than in fibrin or plasma clot. A mixed cell population isolated from Matrigel cultures formed a monolayer in collagen or fibrin-coated dishes but rapidly reorganized into a polygonal network when plated on Matrigel. The observation that gels composed of basement membrane molecules modulate the canalization, proliferation, and organization into networks of vasoformative endothelial cells in three-dimensional cultures supports the hypothesis that the basement membrane is a potent regulator of microvascular growth and morphogenesis. This work was supported by grants from the W. W. Smith Charitable Trust and grants CA14137 and HL43392 from the National Institutes of Health, Bethesda, MD.     

GROWTH PATTERN,REPRODUCTION, AND SELF-THINNING IN SEAWEEDS1

In unispecific plant stands, the logarithm of mean individual weight (w) depends on the logarithm of density (d) by the -3/4 power law (a slope of -1.5 and an intercept ranging from 2.3 to 5.0). The analysis of the w and d relationships in whole cohorts of two seaweed species from the Strait of Gibraltar shows deviations from the canonical equation. The kelp Phyllariopsis purpurascens (C. Agardh) Henry et South (Phaeophyta) growing at a 30-m depth has the lowest intercept value (0.6) recorded for any plant species and a slope not significantly different from -1.5. The slope value is in accordance with those found in species whose growth is not stopped by reproduction. Irradiance under a single layer of blades was lower than the photosynthetic light compensation point, and this could be due to overdispersion of the shoots and, in consequence, to the low intercept value of the self-thinning equation. The w to d relationships in Asparagopsis armata Harvey (Rhodophyta) show two different components: no dependence between these two variables (slope not significantly different from 0) at densities < 500 shoots·m^?2, and a slope more negative (-2.1) than proposed by the -3/2 power law at densities > 500 shoots·m^?2. The pattern at high densities could be due to intraspecific competition for light, whereas the slope ～0 at low densities could be related to inhibition of growth by reproduction (cystocarp and carpospore production). Therefore, rather than being considered exceptional, we suggest that a gradient of variability could be expected in the dependence of w on d when specific growth patterns and reproduction are considered.     

Heterotrophic bacteria, activity and bacterial diversity in two coastal lagoons as detected by culture and 16S rRNA genes PCR amplification and partial sequencing

Activity and numbers of heterotrophic bacteria have indicated that, as expected, Prevost Lagoon is more eutrophic than Arcachon Bay. Amplification and sequence analysis of the 16S rRNA genes from DNA samples extracted directly from the environment allow the determination of phylogenetic relationships among members of microbial communities in natural ecosystems without the need for cultivation. Analysis of partial 16S rRNA gene sequences obtained from Stations A and 11 revealed that, in both environments, a relatively large number of clones related to Cytophaga/FlexibacterBacteroides as well as to -Proteobacteria were found. One hundred percent similarity with the sequences of the data bases were not found for any of the more than a hundred clones studied. In fact for most clones maximum similarity was below 95% for the nucleotide series sequenced. Similarity was not higher with any of the sequences found for the 14 isolates (pure cultures) obtained from the same samples. Redundancy, i.e. number of identical sequences, was higher in the samples from Arcachon. In addition, sequences related to representatives of ten major phylogenetic branches of Eubacteria were obtained from Prevost Lagoon, however only five branches were represented by the data from Arcachon. These findings indicate a higher bacterial diversity in Prévost Lagoon.     

Trinuclear iridium and rhodium complexes: the solution to a puzzle involving the multiple coordination possibilities of 1,8-naphthyridine-2-one ligands

The multiple coordination possibilities of 1,8-naphthyridine-2-one (HOnapy) and 5,7-dimethyl-1,8-napthyridine-2-one (HOMe₂napy) ligands allow the synthesis of a variety of tri- di- and mononuclear complexes, showing fluxional behaviour and frequent exchange of the coordinated ML₂ fragments. Thus, reactions of [M₂(μ-OMe)₂(cod)₂] (cod = 1,5-cyclooctadiene) with HOnapy and HOMe₂napy yield the compounds of the general formula [M(μ-OR₂napy) (cod)]_n (M = Ir, R = Me (1a, 1b, H (2); M = Rh, R = Me (3a, 3b). They crystallise as inconvertible yellow (a) and purple/orange (b) forms and also show a puzzling behaviour in solution. X-ray diffraction studies on both forms (3a, 3b) and spectroscopic data reveal that the yellow forms are mononuclear complexes whilst the dark-coloured crystals contain dinuclear complexes. In solution, the nuclearity of the complexes depends on the solvent. In addition both types of complexes are fluxional. The mixed-ligand complexes [M₂(μ-OMe₂napy)₂(CO)₂(cod)] M = Ir (5), Rh (6) have been isolated and characterised; they are found to be intermediates in the synthesis of the trinuclear complexes [M₃(μ₃-OMe₂napy)₂(CO)₂(cod)₂]⁺ M = Rh (8), Ir (9). Reactions of [IrCl(CO)₂(NH₂-p-tolyl] with the complexes [Rh(μ-OR₂napy)(diolefin)]_n followed by addition of a poor donor anion is a general one-pot synthesis for the hetertrinuclear complexes [Rh₂Ir(μ₃-OR₂napy)₂(CO)₂(diolefin)₂]⁺ (R=Me, DIOLEFIN = cod (10), tetrafluorobenzo-barrelene (tfbb) (11), 2,5-norbornadiene (nbd) (12); R=H, DIOLEFIN=cod (13)). This synthesis follows a stepwise mechanism from the mononuclear to the trinuclear complexes in which mixed-ligand heterodinuclear complexes are involved as intermediates of the type [(diolefin)Rh(μ-OMe₂napy)₂Ir(CO)₂]. Heteronuclear complexes which possess the core [RhIr₂]³⁺, such as [RhIr₂(μ₃-OR₂napy)₂(CO)₂(cod)₂]BF₄ (R=Me (14), H (15)), result from the reaction of 1 or 2 with [Rh(CO)₂S_x]⁺ (S = solvent). The trinuclear complexes undergo two chemically reversible one-electron oxidation processes. The chemical oxidation of 10, 14 and 9 with silver salts gives the mixed-valence trinuclear radicals [Rh₂Ir(μ₃-OMe₂napy)₂(CO)₂(cod)₂]²⁺ (16), [RhIr₂(μ₃-OMe₂napy)₂(CO)₂(cod)₂]²⁺ (17) and [Ir₃(μ₃-OMe₂napy)₂(CO)₂(cod)₂]²⁺ (18), which have been isolated as the perchlorate and tetrafluoroborate salts. The EPR spectrum of 16 indicates that the unpaired electron is essentially in an orbital delocalised on the metals. The molecular structures of the complexes 3a, 3b, 6, 10b and 16a are described. Crystals of 3a are triclinic, P-1, with a = 9.7393(2), b = 14.0148(4), c = 16.0607(4) Å, α = 88.122(3), β = 83.924(3), γ = 87.038(3)°, Z = 4; 3b crystallises in the Pna2_i orthorhhombic space group, with a = 16.7541(3), B = 11.7500(8), c = 17.7508(7) Å, Z = 4; complex 6 is packed in the monoclinic space group P2_i/c, a = 9.6371(1), b = 11.8054(4), c = 27.2010(9) Å, β = 90.556(4)°, Z = 4; crystals of 10b are monoclinic, P2₁/n, with a = 17.546(7), b = 13.232(6), c = 17.437(8) Å, β = 106.18(1)°, Z = 4; crystals of 16a are triclinic, P-1, with a = 10.318(4), b = 12.562(6), C = 19.308(8) Å, α = 92.12(8), β = 97.65(9), γ = 90.68(5)°, Z = 2. The five different structures show the coordination versatility of the OMe₂napy molecule as ligand, which behaves as a N,N′-chelating (3a), bidentate N,O-donor (3b, 6), or as a tridentate N,N′,O-donor bridging ligand (10b, 16a).     

Phytoplankton biomass and production in shelf waters off NW Spain: spatial and seasonal variability in relation to upwelling

Summary Chlorophyll-a and primary production on the euphotic zone of the N-NW Spanish shelf were studied at 125 stations between 1984 and 1992. Three geographic areas (Cantabrian Sea, Rías Altas and Was Baixas), three bathymetric ranges (20 to 60 m, 60 to 150 m and stations deeper than 200 m), and four oceanographic stages (spring and autumn blooms, summer upwelling, summer stratification and winter mixing) were considered. One of the major sources of variability of chlorophyll and production data was season. Bloom and summer upwelling stages have equivalent mean and maximum values. Average chlorophyll-a concentrations approximately doubled in every step of the increasing productivity sequence: winter mixing — summer stratification — high productivity (upwelling and bloom) stages. Average primary production rates increased only 60% in the described sequence. Mean (± sd) values of chlorophyll-a and primary production rates during the high productivity stages were 59.7 ± 39.5 mg Chl-a m^–2 and 86.9 ± 44.0 mg C m^–2 h^–1, respectively. Significant differences in both chlorophyll and primary production resulted between geographic areas in most stages. Only 27 stations showed the effects of the summer upwelling that affected coastal areas in the Cantabrian Sea and Rías Baixas shelf, but also shelf-break stations in the Rías Altas area. The Rías Baixas area had lower chlorophyll than both the Rías Altas and the Cantabrian Sea areas during spring and autumn blooms, but higher during summer upwelling events. On the contrary, primary production rates were higher in the Rías Baixas area during blooms in spring and autumn. Mid-shelf areas showed the highest chlorophyll concentrations during high productivity stages, probably due to the existence of frontal zones in all geographic areas considered. The estimated phytoplankton growth rates were comparable to those of other coastal upwelling systems, with average values lower than the maximum potential growth rates. Doubling rates for upwelling and stratification stages in the northern and Rías Altas shelf areas were equivalent, despite larger biomass accumulations during upwelling events. Low turnover rates of the existing biomass in the Rías Baixas shelf in upwelling stages suggests that the accumulation of phytoplankton was due mainly to the export from the highly productive rías, while the contribution of in situ production to these accumulations was relatively lower.