Purification,crystallization, and preliminary X-ray analysis of PepX,an X-prolyl dipeptidyl aminopeptidase from Lactococcus lactis

The X-prolyl dipeptidyl aminopeptidase PepX, a serine peptidase isolated originally from Lactococcus lactis subsp lactis NCDO 763, was cloned and overproduced in Escherichia coli. The enzyme was isolated in its active form in two purification steps. Crystals of PepX were grown by the hanging drop vapor diffusion method using polyethyleneglycol 4000 as precipitant at pH 5.0. The crystals are orthorhombic with cell dimensions a = 92.8 Å, b = 102.6 Å, and c = 101.6 Å, space group P2₁2₁2, and probably contain one monomer of 87.5 kDa in the asymmetric unit. The crystals, very stable under X-rays, diffract to at least 2.2 Å and are suitable for high-resolution structural analysis. © 1995 Wiley-Liss, Inc.     

Photosynthesis and inorganic carbon acquisition in the cyanobacterium Chlorogloeopsis sp. ATCC 27193

The ability of the morphologically complex cyanobacterium Chlorogloeopsis sp. ATCC 27193 to actively transport and accumulate inorganic carbon (C₁= CO₂+ HCO₃^?+ CO₃^2?) for photosynthetic CO₂ fixation was investigated. Mass-spectrometric assays revealed that Chlorogloeopsis cells grown under C₁ limitation rapidly took up CO₂ from the medium in a light-dependent reaction which was independent of CO₂ fixation. Ethoxyzolamide, a carbonic anhydrase (CA) inhibitor, inhibited CO₂ transport. Since electrometric and mass-spectrometric assays did not detect the presence of a periplasmic CA, it is suggested that CO₂ transport was mediated by a CA-like activity which converted CO₂ to HCO₃^? during passage across the membrane. Radiochemical assays, using H¹⁴CO₃ as substrate, showed that C₃-limited cells also had a high affinity (K_0.5 HCO₃^?= 37 μM), Na⁺-independent HCO₃^? uptake mechanism. HCO₃^?uptake was light dependent and occurred against its electrochemical potential indicating a carrier-mediated, active transport process. The rate of Na⁺-independent HCO₃^? transport was sufficient to account for the steady state rate of CO₂ fixation. Although not absolutely required. Na⁺ did specifically enhance the rate of HCO₃^? transport by up to 2-fold, but had no effect on the apparent affinity of the transport system for HCO₃^? Combined CO₂ and HCO₃^? transport resulted in C₁ accumulation as high as 25 mM and in excess of 300 times the external concentration. The C₁ pool was the source of CO₂ for photo-synthetic fixation and was generated, presumably, by the dehydration of HCO₃^? catalyzed by an intracellular CA. The collective evidence indicates that Chlorogloeopsis has a physiologically functional CO₂-concentrating mechanism which is essential for photosynthesis.     

Energetics of Isolated Hepatocyte Swelling Induced by Sodium Co-transported Amino Acids

This study was designed to investigate the energetics of isolated rat hepatocyte swelling due to sodium-cotransported amino acid accumulation in a medium containing either glucose or octanoate as basal substrate. We show that the size of the increase in cytosolic volume is directly correlated with the total amino acid accumulation, which depends on the difference of electrical potential across the plasma membrane. Such a change in cell volume, with either glucose or octanoate, does not modify the mitochondrial volume. Addition of sodium-cotransported amino acids for which the metabolism was avoided showed that the rise in cell volume, per se, did not change the respiratory rate, p, or phosphate potential in either mitochondrial or cytosolic compartments. Conversely, the large increase in oxidative phosphorylation flux was due to the metabolism of amino acids as a consequence of a rise in electron supply for the respiratory chain rather than an increase in cellular ATP demand, as indicated by the increase in cytosolic phosphate potential. Moreover, although we confirm that octanoate addition largely increases the respiratory rate by a process different from uncoupling, we observed that the same overall thermodynamic driving force through the respiratory chain and the same mitochondrial or cytosolic phosphate potential were maintained for much higher oxygen consumption when octanoate was present. We propose that these octanoate effects are due to a decrease in the actual protons/2 electrons stoichiometry as a consequence of a shift in electron supply toward a two-coupling site instead of a three-coupling site. The change in the FADH₂/NADH formation flux ratio in either fatty acid or carbohydrate oxidation explains such results.     

Passive sensitization of human airways induces myogenic contractile responses in vitro

Mitchell, R. W., K. F. Rabe, H. Magnussen, and A. R. Leff.Passive sensitization of human airways induces myogenic contractile responses in vitro. J. Appl.Physiol. 83(4): 1276-1281, 1997.We assessedeffects of passive sensitization on human bronchial smooth muscle (BSM)response to mechanical stretching in vitro. Bronchial rings were sham(control) or passively sensitized overnight by using sera from donorsdemonstrating sensitivity to Dermatophagoides farinae and having immunoglobulin E (IgE)concentrations of 2,600 ± 200 U/ml. Tissues were fixedisometrically to force transducers to measure responses to electricalfield stimulation (EFS) and quick stretch (QS). The myogenic responseto QS was normalized to the maximal response to EFS (%EFS). Themyogenic response of sensitized BSM was 47.9 ± 10.9 %EFS to a QSof ~6.5% optimal length (L_o);sham-sensitized tissues had a myogenic response of 13.5 ± 6.4 %EFS(P = 0.012 vs. passively sensitized).A QS of ~13% L_o in sensitizedBSM caused a response of 82.8 ± 20.9 %EFS; sham-sensitized tissuesdeveloped a response of 38.2 ± 17.3 %EFS(P = 0.004). BSM incubated with serumfrom nonallergic donors did not demonstrate increased QS response (4.6 ± 1.4 %EFS, P = not significantvs. tissue exposed to atopic sera). However, tissues incubated in serafrom nonatopic donors supplemented with hapten-specific chimeric IgE(JW8) demonstrated augmented myogenic response to QS of ~6.5% L_o (21.9 ± 6.2 %EFS, P = 0.027 vs. nonatopicsera alone). We demonstrate that passive sensitization of human BSMpreparations causes induction and augmentation of myogenic contractionsto QS; this hyperresponsiveness corresponds to the IgE concentration insensitizing sera.

     

Study ofO-sialylation of glycoproteins in C6 glioma cells treated with retinoic acid

When treated with retinoic acidin vivo, C6 glioma cells show an enhancement of CMP-Neu5Ac:Gal 1–3 GalNAc-R -2,3 sialyltransferase activity. A 300kDa glycoprotein was detected by lectin affinoblotting in retinoic acid-treated C6 cells which stained weakly or not at all in control cells. Comparative studies with different lectins demonstrated that this glycoprotein contains 2,3 Neu5Ac Gal-GalNAc O-glycan moieties. Cultures in the presence of an inhibitor of O-glycan synthesis (N-acetylgalactosaminide -O-benzyl) demonstrated that enhancement of staining of the 300 kDa glycoprotein was not due to the increase of the 2,3 sialyltransferase but to thede novo synthesis of the polypeptide chain of this glycoprotein.Abbreviations RA retinoic acid - Neu5Ac N-acetylneuraminic acid - CMP-Neu5Ac cytidine 5 monophosphosialate - 2,3 ST CMP-Neu5Ac:Gal 1–3 GalNAc-R -2,3 sialyltransferase - GalNAc-O-benzyl N-acetylgalactosaminide -O-benzyl - Gal1-3GalNAc-O-benzyl Galactosyl 1-3N-acetylgalactosaminide -O-benzyl - TBS Tris-HCl buffer 50mm pH 7.5 containing NaCl 0.15m and Tween 20 0.05% - B1 buffer TBS containing MgCl₂ 1mm, MnCl₂ 1mm and CaCl₂ 1mm     

Procedures for the Isolation of Cyanobacterial Random Mutants with a Digital Imaging Spectrophotometer

We investigated the use of the Digital Imaging Spectrophotometer (Youvan et al., 1995) for the primary isolation of photosynthetic mutants in the cyanobacterium Synechocystis sp. PCC 6803. We tested the system with two previously characterized mutants of Synechocystis sp. PCC 6803: the Del-1 mutant, a partial deletion mutant of the psbB gene (Eaton-Rye and Vermaas, 1991), and the psbO mutant, a complete deletion of the psbO gene (Burnap and Sherman, 1991). We found that the considiration of colony sizes vs camera resolution is important for avoiding the isolation of false positive mutants. We modified the instrument by adding a magnifying lens for fluorescence imaging of plates inside the sphere. We proposed three ways in which the DIS can be used to isolate cyanobacterial random mutants: direct fluorescence intensity, fluorescence image ratios, and PC/Chl ratios calculated from absorbance. The reliabilty of each of those methods is excellent for differentiating existing PSII deletion mutants. We also proposed a statistical criterion for selecting significantly different mutants.     

Nests of subterranean fungus-growing termites (Isoptera, Macrotermitinae) as nutrient patches for grasses in savannah ecosystems

Savannah are ecosystems in which mineral nitrogen is considered as a limiting factor for plant productivity. They are heterogeneous and spatially structured in patches, or islands, where mineral nitrogen content is concentrated. Among the soil macrofauna, termites of the Macrotermitinae subfamily are major determinants of soil heterogeneity through the biogenic underground nest structures (fungus‐comb chambers) they produce. To study the role of the heterogeneity created by termites on Pennisetum pedicellatum, an herbaceous grass species was grown in greenhouse. This was carried out using an homogeneous soil poor in mineral nitrogen, and an heterogeneous soil with patch, made of (i) Ancistrotermes cavithorax fungus‐comb chamber wall and (ii) soil with the same mineral nitrogen content as the termite handled soil. Plants exhibited a better growth on patch of termite‐modified soil whereas no significant differences were shown with the supply of mineral nitrogen. The presence of fungus‐comb chamber wall material resulted in an increase of fine root biomass and root/shoot ratio. We conclude that termites, through their building activities, may create nutrient patches available to grasses. Concurrently, our data illustrate that the higher mineral nitrogen content in termite‐built structures is not the only factor responsible for plant growth.     

Social Network and Vulnerability: A Clear Link in Bedouin Society (Egypt)

In the last 60 years, the livelihoods of agro-pastoral and pastoral families in the arid and semi-arid zones in North Africa and the Middle East have undergone major changes caused by significant incentives to adopt a sedentary lifestyle and the increasing intensity of drought events. Such changes have also been influenced by land reclamation projects accompanied by the construction of reservoirs and dikes in the dry lands as well as the extension of irrigation canals in the desert in the Coastal Zone of the Western Desert, Egypt. To understand the changes in the traditional social organization of this desert society, and how these social changes have affected families’ ability to adapt to external shocks such as the recent 15-year drought, we developed a typological approach to investigate the link between family livelihoods and social capital in Bedouin society. We showed a clear link between physical assets (mainly land and animals), the nature and intensity of social links within the traditional society, and level of education. The analysis revealed also some new wealth accumulation processes in link with the socio-political influence of urban zones and the increasing level of education in the zone.