Novel biosynthetic functions of lipopolysaccharide rfaJ homologs from Helicobacter pylori

Activity screening and insertional inactivation of lipopolysaccharide (LPS) biosynthetic genes in Helicobacter pylori have led to the successful characterization of two key enzymes encoded by HP0159 (JHP0147) and HP1105 (JHP1032) open reading frames (ORFs) which are members of the large and diverse carbohydrate active enzymes (CAZY) GT-8 (rfaJ) family of glycosyltransferases. Activity screening of a genomic library led to the identification of the enzyme involved in the biosynthesis of the type 2 N-acetyl-lactosamine O-chain backbone, the beta-1,3-N-acetyl-glucosaminyl transferase. In addition, the activity screening approach led to the identification and characterization of a key core biosynthetic enzyme responsible for the biosynthesis of the alpha-1,6-glucan polymer. This alpha-1,6-glucosyltransferase protein is encoded by the HP0159 ORF. Both enzymes play an integral part in the biosynthesis of LPS, and insertional inactivation leads to the production of a truncated LPS molecule on the bacterial cell surface. The LPS structures were determined by mass spectrometry and chemical analyses. The linkage specificity of each glycosyltransferase was determined by nuclear magnetic resonance (NMR) analysis of model compounds synthesized in vitro. A cryogenic probe was used to structurally characterize nanomole amounts of the product of the HP1105 (JHP1032) enzyme. In contrast to the HP0159 enzyme, which displays the GT-8-predicted retaining stereochemistry for the reaction product, HP1105 (JHP1032) is the first member of this GT-8 family to have been shown to have an inverting stereochemistry in its reaction products.     

Influence of divalent cations on the catalytic properties and secondary structure of unadenylylated glutamine synthetase from Azospirillum brasilense

Fully unadenylylated glutamine synthetase (GS) from the endophytic bacterium Azospirillum brasilense Sp245 was isolated and purified. The enzyme was electrophoretically homogeneous and contained strongly bound metal ions, which could not be removed by dialysis. Mn²⁺, Mg²⁺, and Co²⁺ were found to be effective in supporting biosynthetic activity of the A. brasilense GS. Some kinetic properties of Mn²⁺-activated and Mg²⁺-activated unadenylylated GS were characterized. Circular dichroism analysis of the enzyme showed that the A. brasilense GS is a highly structured protein: 59% of its residues form -helices and 13% -strands. Removal of the metal ions from the A. brasilense GS by treatment with EDTA resulted in alterations in the enzyme secondary structure.     

Nonsteroidal anti-inflammatory drugs lower Abeta42 and change presenilin 1 conformation

Recent reports suggest that some commonly used nonsteroidal anti-inflammatory drugs (NSAIDs) unexpectedly shift the cleavage products of amyloid precursor protein (APP) to shorter, less fibrillogenic forms, although the underlying mechanism remains unknown. We now demonstrate, using a fluorescence resonance energy transfer method, that Abeta(42)-lowering NSAIDs specifically affect the proximity between APP and presenilin 1 and alter presenilin 1 conformation both in vitro and in vivo, suggesting a novel allosteric mechanism of action.     

A mathematical model of the interactions in the mixed culture of invertebrates and algae in the “producer–consumer” aquatic biotic cycle

This paper presents a mathematical model of interactions between two herbivorous invertebrates (ciliate Paramecium caudatum and rotifer Brachionus plicatilis) and two planktonic algae (Chlorella vulgaris and Scenedesmus quadricauda) spatially segregated in two compartments of a chemostat–type experimental microcosm system. The model mimics a producer–consumer aquatic biotic cycle, describing the dynamics of the mixed culture of ciliates and rotifers, as consumer compartment, feeding on the mixed algal culture, as producer compartment, under N-limiting conditions. We experimentally found that metabolites of the alga Scenedesmus produce an adverse effect on the reproduction of ciliate Paramecium. Taking this effect into account improved the behavior of the model, the results of which came into qualitative agreement with the experimental results. Both our experimental and modeling approaches demonstrated that, even in conditions of a spatially–segregated producer-consumer biotic cycle, species coexistence is impossible either in the mixed algal culture or in the mixed invertebrate culture. Scenedesmus excluded Chlorella, whereas Brachionus excluded Paramecium.     

A homeostatic mechanism counteracting K(+) -evoked choline release in adult brain

Choline (Ch) is an essential nutrient as the biosynthetic precursor of acetylcholine (ACh) and phospholipids. Under resting conditions, the intracellular accumulation of Ch (above 10-fold), which is positively charged, is governed by the membrane potential and follows the Nernst equation. Accordingly, in synaptosomes from adult rats during depolarization, we observed a linear relationship between release of free cytoplasmic Ch and KCl concentration (2.7-120 mm). The K(+) -evoked Ch release was Ca(2+) -independent and did not originate from ACh or phospholipid hydrolysis. In superfused brain slices of adult rats, however, a K(+) -induced Ch efflux was absent. Also, under in vivo conditions, 30-60 mm KCl failed to increase the extracellular Ch level as shown by microdialysis in adult rat hippocampus. On the contrary, in brain slices from 1-week-old rats, high K(+) as well as 4-aminopyridine evoked a marked Ch efflux in a concentration-dependent fashion. This phenomenon faded within 1 week. Hemicholinium-3 (HC-3, 1 and 10 microm), a blocker of cellular choline uptake, caused a marked efflux of choline from adult rat slices but no or significantly less release from immature slices. We conclude that depolarization of synaptic endings causes a Ca(2+) -independent release of free cytoplasmic Ch into the extracellular space. In adult rat brain, this elevation of Ch is counteracted by a homeostatic mechanism such as uptake into brain cells.     

Astrocyte dystrophy in ageing brain parallels impaired synaptic plasticity

Little is known about age‐dependent changes in structure and function of astrocytes and of the impact of these on the cognitive decline in the senescent brain. The prevalent view on the age‐dependent increase in reactive astrogliosis and astrocytic hypertrophy requires scrutiny and detailed analysis. Using two‐photon microscopy in conjunction with 3D reconstruction, Sholl and volume fraction analysis, we demonstrate a significant reduction in the number and the length of astrocytic processes, in astrocytic territorial domains and in astrocyte‐to‐astrocyte coupling in the aged brain. Probing physiology of astrocytes with patch clamp, and Ca²⁺ imaging revealed deficits in K⁺ and glutamate clearance and spatiotemporal reorganisation of Ca²⁺ events in old astrocytes. These changes paralleled impaired synaptic long‐term potentiation (LTP) in hippocampal CA1 in old mice. Our findings may explain the astroglial mechanisms of age‐dependent decline in learning and memory.     

On the Estimation of Time Dependent Lift of a European Starling (Sturnus vulgaris) during Flapping Flight

We study the role of unsteady lift in the context of flapping wing bird flight. Both aerodynamicists and biologists have attempted to address this subject, yet it seems that the contribution of unsteady lift still holds many open questions. The current study deals with the estimation of unsteady aerodynamic forces on a freely flying bird through analysis of wingbeat kinematics and near wake flow measurements using time resolved particle image velocimetry. The aerodynamic forces are obtained through two approaches, the unsteady thin airfoil theory and using the momentum equation for viscous flows. The unsteady lift is comprised of circulatory and non-circulatory components. Both approaches are presented over the duration of wingbeat cycles. Using long-time sampling data, several wingbeat cycles have been analyzed in order to cover both the downstroke and upstroke phases. It appears that the unsteady lift varies over the wingbeat cycle emphasizing its contribution to the total lift and its role in power estimations. It is suggested that the circulatory lift component cannot assumed to be negligible and should be considered when estimating lift or power of birds in flapping motion.