Improved high-performance liquid chromatographic separation of peptidoglycan isolated from various Staphylococcus aureus strains for mass spectrometric characterization

Reversed-phase high-performance liquid chromatography (RP-HPLC) of muropeptides, obtained by muramidase digestion of peptidoglycan in combination with amino acid analysis and plasma desorption time-of-flight mass spectrometry is today by far the best tool to analyze the fine structure of the peptidoglycans. Here we report further improvements of the RP-HPLC separation of muropeptides for analyzing the peptidoglycans of various methicillin-resistant strains of Staphylococcus aureus, with emphasis on a more detailed characterization of the interpeptide bridge of the peptidoglycans of this species.     

Amyloid induced suicidal erythrocyte death.

Amyloid peptides are known to induce apoptosis in a wide variety of cells. Erythrocytes may similarly undergo suicidal death or eryptosis, which is characterized by scrambling of the cell membrane with subsequent exposure of phosphatidylserine (PS) at the cell surface. Eryptosis is triggered by increase of cytosolic Ca(2+) activity and by activation of acid sphingomyelinase with subsequent formation of ceramide. Triggers of eryptosis include energy depletion and isosmotic cell shrinkage (replacement of extracellular Cl(-) by impermeable gluconate for 24 h). The present study explored whether amyloid peptide Abeta (1-42) could trigger eryptosis and to possibly identify underlying mechanisms. Erythrocytes from healthy volunteers were exposed to amyloid and PS-exposure (annexin V binding), cell volume (forward scatter), cytosolic Ca(2+) activity (Fluo3 fluorescence) and ceramide formation (anti-ceramide antibody) were determined by FACS analysis. Exposure of erythrocytes to the amyloid peptide Abeta (1-42) (> or = 0.5 microM) for 24 h significantly triggered annexin V binding, an effect mimicked to a lesser extent by the amyloid peptide Abeta (1-40) (1 microM). Abeta (1-42) (> or = 1.0 microM) further significantly decreased forward scatter of erythrocytes. The effect of Abeta (1-42) (> or = 0.5 microM) on erythrocyte annexin V binding was paralleled by formation of ceramide but not by significant increase of cytosolic Ca(2+) activity. The presence of Abeta (1-42) further significantly enhanced the eryptosis following Cl(-) depletion but not of glucose depletion for 24 hours. The present observations disclose a novel action of Abeta (1-42), which may well contribute to the pathophysiological effects of amyloid peptides, such as vascular complications in Alzheimer's disease.     

Parturition in horses is dominated by parasympathetic activity of the autonomous nervous system

External and internal stressors prolong parturition in different species. At parturition, sympathoadrenal activation should be avoided because an increased sympathetic tone may cause uterine atonia via β₂-receptors. We hypothesized that at physiological parturition, horses are under parasympathetic dominance, and stress-response mechanisms are not activated during delivery of the foal. To evaluate stress responses, heart rate, heart rate variability, catecholamines, and cortisol were analyzed in mares (n = 17) throughout foaling. Heart rate decreased from 2 hours before (51 ± 1 beats/minute) to 2 hours after delivery (41 ± 2 beats/minute; P < 0.05). Heart rate variability variables, standard deviation of the beat-to-beat interval, and root mean square of successive beat-to-beat differences, changed over time (P < 0.05) with the highest values within 15 minutes after delivery. The number of mares with atrioventricular blocks and the number of atrioventricular blocks per mare increased over time (P < 0.01) and were significantly elevated from 15 minutes before to 45 minutes after birth of the foal. Salivary cortisol concentrations increased to a maximum at 30 minutes after delivery (25.0 ± 3.4 ng/mL; P < 0.01). Plasma epinephrine and norepinephrine concentrations showed significant fluctuations from rupture of the allantochorion to expulsion of the fetal membranes (P < 0.01) but were not markedly elevated at any time. In conclusion, mares give birth under high parasympathetic tone. Cortisol release during and after foaling is most likely part of the endocrine pathways regulating parturition and not a labor-associated stress response.     

Similar qualitative and quantitative changes of mitochondrial respiration following strength and endurance training in normoxia and hypoxia in sedentary humans

Endurance and strength training are established as distinct exercise modalities, increasing either mitochondrial density or myofibrillar units. Recent research, however, suggests that mitochondrial biogenesis is stimulated by both training modalities. To test the training "specificity" hypothesis, mitochondrial respiration was studied in permeabilized muscle fibers from 25 sedentary adults after endurance (ET) or strength training (ST) in normoxia or hypoxia [fraction of inspired oxygen (Fi(O(2))) = 21% or 13.5%]. Biopsies were taken from the musculus vastus lateralis, and cycle-ergometric incremental maximum oxygen uptake (VO(2max)) exercise tests were performed under normoxia, before and after the 10-wk training program. The main finding was a significant increase (P < 0.05) of fatty acid oxidation capacity per muscle mass, after endurance and strength training under normoxia [2.6- and 2.4-fold for endurance training normoxia group (ET(N)) and strength training normoxia group (ST(N)); n = 8 and 3] and hypoxia [2.0-fold for the endurance training hypoxia group (ET(H)) and strength training hypoxia group (ST(H)); n = 7 and 7], and higher coupling control of oxidative phosphorylation. The enhanced lipid oxidative phosphorylation (OXPHOS) capacity was mainly (87%) due to qualitative mitochondrial changes increasing the relative capacity for fatty acid oxidation (P < 0.01). Mitochondrial tissue-density contributed to a smaller extent (13%), reflected by the gain in muscle mass-specific respiratory capacity with a physiological substrate cocktail (glutamate, malate, succinate, and octanoylcarnitine). No significant increase was observed in mitochondrial DNA (mtDNA) content. Physiological OXPHOS capacity increased significantly in ET(N) (P < 0.01), with the same trend in ET(H) and ST(H) (P < 0.1). The limitation of flux by the phosphorylation system was diminished after training. Importantly, key mitochondrial adaptations were similar after endurance and strength training, regardless of normoxic or hypoxic exercise. The transition from a sedentary to an active lifestyle induced muscular changes of mitochondrial quality representative of mitochondrial health.     

Physiologische Untersuchungen an einer ungewöhnlich säureresistenten Chlorella

Summary The influence of the hydrogen-ion concentration on the growth and metabolism of a highly acid-resistant green alga, Chlorella ellipsoidea (strain Marburg St), was studied. Chlorella pyrenoidosa (Emerson strain) served as a normal control organism. Growth of Chlorella ellipsoidea occurs in the entire range from Ph 2.0 to Ph 10, whereas for Chlorella pyrenoidosa the limits were found to be Ph 3.5 and Ph 10. Respiration is much less sensitive to hydrogen-ion concentration in the acid-resistant as compared to the normal strain. Thus an increase in acidity from Ph 4.0 to Ph 2.0 increases the respiratory oxygen uptake by 120% in Chlorella pyrenoidosa and by 25% in Chlorella ellipsoidea. In addition, only the less resistant Chlorella pyrenoidosa shows an accumulation of nitrite in the dark in acid culture media, indicating a disturbance of the normal course of nitrate reduction under these conditions. On the other hand, the rate of photosynthesis of both organisms was found to be almost independent of acidity between Ph 4.0 and Ph 2.0. At the acid and alkaline limits of growth in both algae, an inhibition of cell division leads to an increase of cell size and dry weight per cell, frequently connected with the occurrence of bizarre giant cells. — In addition, adaptation phenomena were found to play a role in determining the acid limit of growth. Cells of Chlorella ellipsoidea, after inoculation from normal medium (Ph about 6) into a solution of Ph 2.0, begin growth at a high rate only after a lag of about two weeks. Cells grown previously in an acid medium, however, immediately resume growth upon inoculation into a medium of Ph 2.0. This adaptation involves a considerable reduction of cell size.     

Conjugative transfer functions of broad-host-range plasmid RK2 are coregulated with vegetative replication

The kilB locus (which is unclonable in the absence of korB) of broad-host-range plasmid RK2 (60 kb) lies between the trfA operon (co-ordinates 16.4 to 18.2 kb), which encodes a protein essential for vegetative replication, and the Tra2 block of conjugative transfer genes (co-ordinates 20.0 to 27.0 kb). Promoter probe studies indicated that kilB is transcribed clockwise from a region containing closely spaced divergent promoters, one of which is the trfA promoter. The repression of both promoters by korB suggested that kilB may also play a role in stable maintenance of RK2. We have sequenced the region containing kilB and analysed it by deletion and insertion mutagenesis. Loss of the KilB+ phenotype does not result in decreased stability of mini RK2 plasmids. However insertion in ORFI (kilBI) of the region analysed results in a Tra- phenotype in plasmids which are otherwise competent for transfer, demonstrating that this locus is essential for transfer and is probably the first gene of the Tra2 region. From the kilBI DNA sequence KilBI is predicted to be 34995 Da, in line with M(r) = 36,000 observed by sodium dodecyl sulphate/polyacrylamide gel electrophoresis, and contains a type I ATP-binding motif. The purified product was used to raise antibody which allowed the level of KilBI produced from RK2 to be estimated at approximately 2000 molecules per bacterium. Protein sequence comparisons showed the highest homology score with VirB11, which is essential for the transfer of the Agrobacterium tumefaciens Ti plasmid DNA from bacteria to plant cells. The sequence similarity of both KilBI and VirB11 to a family of protein export functions suggested that KilBI may be involved in assembly of the surface-associated Tra functions. The data presented in this paper provide the first demonstration of coregulation of genes required for vegetative replication and conjugative transfer on a bacterial plasmid.     

Seed banks on Attalea phalerata (Arecaceae) stems in the Pantanal wetland, Brazil

Background and Aims

Seeds can accumulate in the soil or elsewhere, such as on the stems of palms when these are covered by persistent sheaths. These sheaths could act as a safe site for some species. Here, we studied whether persistent sheaths of the palm Attalea phalerata (Arecaceae) are available sites for seed accumulation in the Pantanal wetland of Brazil. We also investigated whether the composition, richness and diversity of species of seeds in the persistent sheaths are determined by habitat (riparian forest and forest patches) and/or season (wet and dry).

Methods

All accumulated material was collected from ten persistent sheaths along the stems of 64 A. phalerata individuals (16 per habitat and 16 per season). The material was then individually inspected under a stereomicroscope to record seed species and number.

Key Results

Of the 640 sheaths sampled, 65 % contained seeds (n = 3468). This seed bank included 75 species belonging to 12 families, and was primarily composed of small, endozoochoric seeds, with a few abundant species (Cecropia pachystachya and Ficus pertusa). Moraceae was the richest family (four species) and Urticaceae the most abundant (1594 seeds). Stems of A. phalerata in the riparian forest had 1·8 times more seeds and 1·3 times more species than those in forest patches. In the wet season we sampled 4·1 times more seeds and 2·2 more species on palm stems than in the dry season. Richness did not differ between habitats, but was higher in the wet season. Abundance was higher in forest patches and in the wet season.

Conclusions

Attalea phalerata stems contain a rich seed bank, comparable to soil seed banks of tropical forests. As most of these seeds are not adapted to grow in flooding conditions, palm stems might be regarded as safe sites for seeds (and seedlings) to escape from the seasonal flooding of the Pantanal.     

Specialized 16SrX phytoplasmas induce diverse morphological and physiological changes in their respective fruit crops

The host-pathogen combinations—Malus domestica (apple)/`Candidatus Phytoplasma mali´, Prunus persica (peach)/`Ca. P. prunorum´ and Pyrus communis (pear)/`Ca. P. pyri´ show different courses of diseases although the phytoplasma strains belong to the same 16SrX group. While infected apple trees can survive for decades, peach and pear trees die within weeks to few years. To this date, neither morphological nor physiological differences caused by phytoplasmas have been studied in these host plants. In this study, phytoplasma-induced morphological changes of the vascular system as well as physiological changes of the phloem sap and leaf phytohormones were analysed and compared with non-infected plants. Unlike peach and pear, infected apple trees showed substantial reductions in leaf and vascular area, affecting phloem mass flow. In contrast, in infected pear mass flow and physicochemical characteristics of phloem sap increased. Additionally, an increased callose deposition was detected in pear and peach leaves but not in apple trees in response to phytoplasma infection. The phytohormone levels in pear were not affected by an infection, while in apple and peach trees concentrations of defence- and stress-related phytohormones were increased. Compared with peach and pear trees, data from apple suggest that the long-lasting morphological adaptations in the vascular system, which likely cause reduced sap flow, triggers the ability of apple trees to survive phytoplasma infection. Some phytohormone-mediated defences might support the tolerance.