Molecular Diversity and Evolution of bla TEM Genes Encoding β-Lactamases Resistant to Clavulanic Acid in Clinical E. coli

The molecular diversity of inhibitor-resistant TEM (IRT) enzymes was explored using a strategy which involved DNA amplification by polymerase chain reaction (PCR), analysis of restriction fragment length polymorphism (RFLP), and direct nucleotide sequencing. The study of plasmid-borne genes from 27 strains, resistant to amoxicillin and β-lactamase-inhibitor combinations, identified mutations resulting in amino acid change at positions 69, 244, 275, and 276 known to be associated with the IRT phenotype and a mutation at nucleotide position 162 in the promoter region. These mutations were found to lie on two different gene sequences, described here as ``TEM-1B like' and ``TEM-2 like' restriction linkage groups. Further analysis, of nucleotide sequences of promoter and coding regions of the β-lactamases, confirmed that a given mutation causing IRT phenotype could be associated with two different gene sequence frameworks and two different causal mutations could lie on identical gene sequence framework. These data argue in favor of convergent phenotypic evolution of IRT enzymes under the selective pressure imposed by the intensive clinical use of β-lactam–β-lactamase inhibitor combinations. Received: 18 March 1996 / Accepted: 15 July 1996     

Muscarinic Acetylcholine Receptor Enhances Phosphatidylcholine Hydrolysis via Phospholipase D in Bovine Chromaffin Cells in Culture

Although it is well-established that inositol-containing lipids serve as precursors of intracellular second messenger molecules in chromaffin cells, we describe some findings that show the formation of diacylglycerol from phosphatidylcholine in response to agonist-mediated stimulation. Stimulation of chromaffin cells by acetylcholine produced a high turnover of phosphatidylcholine, as suggested by the release of [3H]choline derived from [3H]-phosphatidylcholine in experiments performed with [3H]choline chloride-prelabeled cells. An enhanced breakdown of phosphatidylcholine was also inferred from the finding of an increased formation of [3H]diacylglycerol in chromaffin cells prelabeled with [3H]glycerol. The diacylglycerol mass that accumulated after stimulation showed a distinct temporal course and seemed to exceed the mass that has been reported to be derived from phosphatidylinositol. In keeping with the purported origin from phosphatidylcholine, diacylglycerol showed a high content in [3H]oleate molecular species. Phospholipase D activity measurements and experiments performed in the presence of propranolol (an inhibitor of phosphatidic acid:phosphohydrolase) suggested that phosphatidylcholine is hydrolyzed by a phospholipase D activity, producing phosphatidic acid, which is subsequently degraded to diacylglycerol, rather than by a phospholipase C. Incubation of chromaffin cells in the presence of atropine before addition of acetylcholine showed complete inhibition of the increased formation of [3H]-diacylglycerol, whereas d-tubocurarine failed to do so. Taken together, these results suggest that acetylcholine activates phosphatidylcholine breakdown and diacylglycerol formation in chromaffin cells via a muscarinic-type receptor.     

Myelin Lipids in the Developing Cerebrum, Cerebellum, and Brain Stem of Normal and Undernourished Children

Abstract: The developmental lipid profiles in the human cerebrum, cerebellum and brain stem are presented, with special reference to galactolipids as myelin markers to trace myelination in the three main parts of the human CNS. A group of undernourished children were also studied to test the vulnerability of myelinogenesis in the different regions of the human brain. Myelination was well advanced in the brain stem with regard to the other brain regions, a fact reflected in the much higher concentration of myelin lipids in the brain stem of the human foetus of 26 weeks of gestational age. The cerebrum, on the other hand, had the lowest galactolipid concentration during the prenatal period, galactolipid levels in the cerebellum being four times higher. From just before the end of gestation the accretion of galactolipids accelerated enormously in the cerebrum, whereas it slowed down considerably in the cerebellum. Consequently, in relation to prenatal levels galactolipids increased most rapidly in the cerebrum, followed by the cerebellum and finally by the brain stem. These regional differences were in clear contrast to data from the rat, as was the finding that only the cerebrum of undernourished children had a galactolipid concentration significantly decreased with respect to normal values. A relationship between the different myelination patterns in the human and the rat and the distinct vulnerability of myelinogenesis in the two species is suggested.     

Ant community composition and functional traits in new grassland strips within agricultural landscapes

1. Ongoing intensification and fragmentation of European agricultural landscapes dramatically reduce biodiversity and associated functions. Enhancing perennial noncrop areas holds great potential to support ecosystem services such as ant‐mediated pest control.
2. To study the potential of newly established grassland strips to enhance ant diversity and associated functions, we used hand collection data and predation experiments to investigate differences in (a) ant community composition and (b) biocontrol‐related functional traits, and (c) natural pest control across habitats in cereal fields, old grasslands, and new grassland transects of three years of age.
3. Ant species diversity was similar between new and old grasslands, but significantly higher in new grasslands than in surrounding cereal fields. Contrary, ant community composition of new grasslands was more similar to cereal fields and distinct from the species pool of old grasslands. The functional trait space covered by the ant communities showed the same distribution between old and new grasslands. Pest control did not differ significantly between habitat types and therefore could not be linked to the prevalence of functional ant traits related to biocontrol services in new grasslands.
4. Our findings not only show trends of convergence between old and new grasslands, but also indicate that enhancing ant diversity through new grasslands takes longer than three years to provide comparable biodiversity and functionality.
5. Synthesis and applications: Newly established grasslands can increase ant species richness and abundance and provide a consistent amount of biocontrol services in agroecosystems. However, three years after their establishment, new grasslands were still dominated by common agrobiont ant species and lacked habitat specialists present in old grasslands, which require a constant supply of food resources and long colony establishment times. New grasslands represent a promising measure for enhancing agricultural landscapes but must be preserved in the longer term to promote biodiversity and resilience of associated ecosystem services.

     

Characterization and amino acid sequence of IRT-4, a novel TEM-type enzyme with a decreased susceptibility to β-lactamase inhibitors

Abstract A novel procedure was used to purify a cytosolic chitinase from Candida albicans to electrophoretic homogeneity. The results represent the first demonstration of the purification of a fungal intracellular chitinase using the criterion of a single band detected following silver-staining of a polyacrylamide gel run under denaturing conditions. Purified chitinase had pH and temperature optima of 5.0 and 50°C, respectively. Inhibition of enzyme activity by allosamidin was pH-dependent occuring maximally at pH 8.0. Phospholipids had similar marked and highly specific effects on the activities of both the purified soluble enzyme and a solubilized microsomal chitinase from C. albicans . Evidence is provided for the existence of a complex chitinolytic system in this organism.     

BCL(X)L and BCL2 increase mitochondrial dynamics in breast cancer cell: Evidence from functional and genetic studies

BCL2 family proteins are important regulators of mitochondrial outer membrane permeabilization (MOMP). In recent years, BCL2 family proteins have also been linked to the regulation of mitochondrial bioenergetics and dynamics. Given their overexpression in breast cancer cells, we sought to explore whether two key members of this family, BCL2 and BCL(X)L impacted on mitochondrial fusion/fission processes. By employing a single cell imaging and RNA sequencing we found that overexpression of BCL2 or BCL(X)L increases mitochondrial dynamics and alters the expression profile of genes involved in this process. Collectively, our data show that overexpression of BCL2 proteins regulates mitochondrial dynamics in breast cancer tumor cells.