A synthetic medium for continuous culture of the S-layer carrying Bacillus stearothermophilus PV 72 and studies on the influence of growth conditions on cell wall properties

Bacterial cell surface layers (S-layers) which show a crystalline structure, defined pores, and a regular arrangement of functioal groups can be used for production of isoporous ultrafiltration membranes and as a matrix for immobilization of macromolecules. S-layer-carrying cell wall fragments from thermophilic Bacillaceae possess an extremely thin peptidoglycan-containing layer with pores larger than those in the S-layer lattice. Thus, they can directly be used for biotechnological applications, when an S-layer protein pool is stored in the rigid cell wall layer which is released during cell wall preparation, forming an inner S-layer. In the present study, a synthetic medium for Bacillus stearothermophilus PV 72 was developed by applying the pulse and shift technique with the aim to produce cell wall fragments with before-mentioned properties by varying the growth conditions in condtinuous culture. The organism was grown at 57 degrees C in a bioreactor with 1 L working volume equipped with exhaust gas analysis and connected to a PC-based process control system. Biomass concentration was 2.2 g/L out of 8 g/L glucose at a dilution rate of 0.3 h(-1), giving a biomass productivity of 0.66 g/L h. Although the organism was grown under different conditions, no change in peptidoglycan composition, extent of peptidoglycan crosslinking, and content of secondary cell wall polymers was observed. The amount of S-layer protein pool stored in the rigid cell wall layer and the autolytic activity depended mainly on the specific growth rate. Cell wall fragments with properties required for ultrafiltration membrane production could be produced by parameter settings in continuous culture. (c) 1995 John Wiley & Sons, Inc.     

Permeability of Xenopus laevis embryos: Specific incorporation of precursors into eukaryotic proteins and nucleic acids

All amino acids and several nucleic acid precursors are taken up by Xenopus laevis embryos. The embryos are completely intact and not modified in any way. These precursors are directly incorporated into the macromolecules of Xenopus embryos and not prokaryotic contaminants as has been previously claimed. Radioactive leucine is incorporated into Xenopus laevis ribosomal proteins as characterized by sucrose gradient centrifugation. The uptake of the amino acids is cycloheximide sensitive and unaffected by chloramphenicol. Radioactive adenosine and orotic acid are taken up and incorporated into tRNA and rRNA at high levels as characterized by sucrose gradients and electrophoresis. These characterizations of labeled macromolecules unequivocally show that normal Xenopus laevis embryos will take up and incorporate labeled precursors to levels which are sufficient to study cellular biochemical events at such early stages of development.     

Substructural specificity and polyvalent carbohydrate recognition by the Entamoeba histolytica and rat hepatic N- acetylgalactosamine/galactose lectins

Both the Entamoeba histolytica lectin, a virulence factor for the causative agent of amebiasis, and the mammalian hepatic lectin bind to N-acetylgalactosamine (GalNAc) and galactose (Gal) nonreducing termini on oligosaccharides, with preference for GalNAc. Polyvalent GalNAc- derivatized neoglycoproteins have >1000-fold enhanced binding affinity for both lectins (Adler,P., Wood,S.J., Lee,Y.C., Lee,R.T., Petri,W.A.,Jr. and Schnaar,R.L.,1995, J. Biol. Chem ., 270, 5164-5171). Substructural specificity studies revealed that the 3-OH and 4-OH groups of GalNAc were required for binding to both lectins, whereas only the E.histolytica lectin required the 6-OH group. Whereas GalNAc binds with 4-fold lower affinity to the E.histolytica lectin than to the mammalian hepatic lectin, galactosamine and N-benzoyl galactosamine bind with higher affinity to the E. histolytica lectin. Therefore, a synthetic scheme for converting polyamine carriers to poly-N-acyl galactosamine derivatives (linked through the galactosamine primary amino group) was developed to test whether such ligands would bind the E.histolytica lectin with high specificity and high affinity. Contrary to expectations, polyvalent derivatives including GalN6lys5, GalN4desmosine, GalN4StarburstTMdendrimer, and GalN8StarburstTMdendrimer demonstrated highly enhanced binding to the mammalian hepatic lectin but little or no enhancement of binding to the E.histolytica lectin. We propose that the mammalian hepatic lectin binds with greatest affinity to GalNAc "miniclusters," which mimic branched termini of N-linked oligosaccharides, whereas the E.histolytica lectin binds most effectively to "maxiclusters," which may mimic more widely spaced GalNAc residues on intestinal mucins.      

Enzyme formation by a yeast cell wall lytic Arthrobacter sp.: formation of \(1,3)-glucanase

The kinetics of \-1,3-glucanase (EC 3.2.1.39; 1,3-\-d-glucan-glucano-hydrolase) formation by a yeast cell wall lytic Arthrobacter species was studied. Yeast glucan as a substrate yielded 360 units (U)/l, but it appeared to be unsuitable for fermentation purposes because of its insolubility and its residual content of glycogen. Growth on water-soluble \(1,3)-glucan [maximum specific growth rate (µ_max)=0.19 h^–1] was governed by different saccharides liberated by enzyme action on glucan. Enzyme formation was repressed by glucose and derepressed by its restricted availability during late exponential and stationary growth. At least 380 U/l of \(1,3)-glucanase were formed. Lactose and lactulose were detected as precursors of potent inducers for \(1,3)-glucanase, the first being a cheap and easily available substrate for large-scale cultivations. Growth rates were reduced (µ_max=0.18 h^–1 and µ_max=0.13 h^–1, respectively), enzyme synthesis occurred only during post-logarithmic growth. The \(1,3)-glucanase levels (260 U/l) formed were comparable to that attained with glucan as a substrate. In continuous culture no enzyme was formed under steady-state conditions but it occurred during transient states after shifting the dilution rate to lower values. Correspondence to: W. Hampel     

Spraydrying of yeast-lytic enzymes from Arthrobacter sp.

Summary Spray drying processes are widely used for large scale preservation of biological materials e.g milk, whey, yeast, egg white, etc. Nevertheless, there is an increasing tendency for drying of catalytic active proteins. The paper presents results from experiments which were carried out to test the applicability of spray drying for the preservation of several bacterial enzymes used for the lysis of yeast cell-walls (-1,3-glucanase, -mannanase, chitinase and amylase) and formed by an Arthrobacter species. Enzyme solutions were obtained by concentrating the cell free cultivation liquid by cross-flow ultrafiltration. Due to the low protein content of the liquid preparation, several substances were added as stabilizers or carriers (eg. sucrose, KCl, MgSO₄) and the effect was studied in further experiments. Spray drying was carried out at inlet air temperatures of 110 – 120°C and outlet air temperatures of 65 – 72°C. Best results were obtained by the addition of 10% KCl, the preparation having a crystalline consistency and a 60% yield in activity.     

PDK1 deficiency in POMC-expressing cells reveals FOXO1-dependent and -independent pathways in control of energy homeostasis and stress response

Insulin- and leptin-stimulated phosphatidylinositol-3 kinase (PI3K) activation has been demonstrated to play a critical role in central control of energy homeostasis. To delineate the importance of pathways downstream of PI3K specifically in pro-opiomelanocortin (POMC) cell regulation, we have generated mice with selective inactivation of 3-phosphoinositide-dependent protein kinase 1 (PDK1) in POMC-expressing cells (PDK1(DeltaPOMC) mice). PDK1(DeltaPOMC) mice initially display hyperphagia, increased body weight, and impaired glucose metabolism caused by reduced hypothalamic POMC expression. On the other hand, PDK1(DeltaPOMC) mice exhibit progressive, severe hypocortisolism caused by loss of POMC-expressing corticotrophs in the pituitary. Expression of a dominant-negative mutant of FOXO1 specifically in POMC cells is sufficient to ameliorate positive energy balance in PDK1(DeltaPOMC) mice but cannot restore regular pituitary function. These results reveal important but differential roles for PDK1 signaling in hypothalamic and pituitary POMC cells in the control of energy homeostasis and stress response.     

Changes in marsh nekton communities along the salinity gradient of the Schelde river, Belgium and The Netherlands

Nekton was sampled in five marshes along the salinity gradient of the Schelde River. The utilisation of three different habitats (large and small creek, marsh pond) by fish and macrocrustacean species was compared among the five sampling sites. In the larger channels fyke nets were deployed to capture fish and macrocrustaceans leaving the marsh at ebb while block nets were set in smaller intertidal creeks. Fish traps passively sampled fish and shrimp in the marsh ponds. The tidal freshwater marsh had a species poor fauna and only a low number of fish was caught. Besides some freshwater species (Alburnoides bipunctatus, Carassius carassius) the European eel, Anguilla anguilla was still present. The four other marshes had a similar community structure although Platichthys flesus was absent from the euhaline area. Among fish species, dominance of Dicentrarchus labrax, Platichthys flesus and Pomatoschistus microps was observed. Carcinus maenas and Palaemonetes varians were the most abundant macrocrustacean species in every marsh. Between the large and small intertidal creeks there was no difference in nekton species composition. The main species used both habitats. Marsh ponds were utilized intensively only by two species, Pomatoschistus microps and Palaemonetes varians in every marsh.     

Antiplatelet and anticoagulant therapy in elective percutaneous coronary intervention

Thrombosis plays a major role in acute vessel closure both after coronary balloon angioplasty and after stenting. This review will address the role of antiplatelet and anticoagulant therapy in preventing early thrombotic complications after percutaneous coronary intervention. The focus will be on agents that are routinely available and commonly used.     

Effects of cadmium and lead stress on somatic embryogenesis of coniferous species. Part II: Changes of thiol substances

Conifers are often used as biomarkers of industrial pollution; however, little is known about the effects of heavy metals on them because only a few species have been tested. The aim of this work was to investigate the effects of cadmium (Cd²⁺) and lead (Pb²⁺) at three different concentrations (50, 250, and 500 µM) on the detoxification potential of Abies alba and Picea abies embryogenic cell masses throughout the 21-day proliferation period. Embryogenic cell masses of A. alba and P. abies responded to treatment with cadmium and lead by inducing phytochelatins and their biosynthetic intermediates. With increasing heavy metal concentrations, glutathione was used for the synthesis of phytochelatins enabling the tissues to bind to heavy metal ions and thereby avoiding the production of reactive oxygen species. Lead in A. alba and cadmium in both species caused similar increases of all antioxidative thiol compounds; thus, similar mechanisms involving a heavy metal-induced stress response can be assumed. In P. abies, the lowest lead concentration tested provoked the highest antioxidative response. Since a very low uptake of lead into the tissue was observed, the higher resistance of P. abies can be attributed to its ability to reduce lead uptake after longer exposure times. The results of cadmium treatment of both species and lead treatment of A. alba indicated the possibility of testing these coniferous species as potential phytoremediators. This is the first study to analyze the effects of heavy metals on the low-molecular-weight plant thiol content in A. alba embryogenic cell masses.     

Young microglia restore amyloid plaque clearance of aged microglia

Alzheimer′s disease (AD) is characterized by deposition of amyloid plaques, neurofibrillary tangles, and neuroinflammation. In order to study microglial contribution to amyloid plaque phagocytosis, we developed a novel ex vivo model by co‐culturing organotypic brain slices from up to 20‐month‐old, amyloid‐bearing AD mouse model (APPPS1) and young, neonatal wild‐type (WT) mice. Surprisingly, co‐culturing resulted in proliferation, recruitment, and clustering of old microglial cells around amyloid plaques and clearance of the plaque halo. Depletion of either old or young microglial cells prevented amyloid plaque clearance, indicating a synergistic effect of both populations. Exposing old microglial cells to conditioned media of young microglia or addition of granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) was sufficient to induce microglial proliferation and reduce amyloid plaque size. Our data suggest that microglial dysfunction in AD may be reversible and their phagocytic ability can be modulated to limit amyloid accumulation. This novel ex vivo model provides a valuable system for identification, screening, and testing of compounds aimed to therapeutically reinforce microglial phagocytosis.