Construction of a reporter plasmid for screening in vivo promoter activity in Francisella tularensis

Francisella tularensis is a facultative intracellular bacterium that survives and multiplies inside macrophages. Here we constructed a new promoter probe plasmid denoted pKK214 by introduction of a promoter-less chloramphenicol acetyltransferase (cat) gene into the shuttle vector pKK202. A promoter library was created in F. tularensis strain LVS by cloning random chromosomal DNA fragments into pKK214. Approximately 15% of the recombinant bacteria showed chloramphenicol resistance in vitro. The promoter library was also used to infect macrophages in the presence of chloramphenicol and after two cycles of infection the library contained essentially only chloramphenicol resistance clones which shows that pKK214 can be used to monitor F. tularensis genes that are expressed during infection.     

Concentrations of Amino Acids in Extracellular Fluid After Opening of the Blood-Brain Barrier by Intracarotid Infusion of Protamine Sulfate

Abstract: This article evaluates the influence of an opening of the blood-brain barrier (BBB) on compounds in brain extracellular fluid. The concentrations of amino acids and some other primary amines were determined in dialysates sampled from the right parietal cortex of rats before and after an intracarotid infusion of protamine sulfate. Extravasated plasma proteins were visualized by Evans blue/albumin and immunohistochemistry. CSF albumin— an indicator of blood-CSF barrier opening—was quantified with immunoelectrophoresis. The brains were macroscopically edematous after 10 mg but not after 5 mg of protamine sulfate. The higher dose led to a 50% death rate. The concentrations of amino acids did not change 10 min after the BBB opening. No significant alterations in the amino acid concentrations were observed after the lower dose. The concentrations of glutamate, aspartate, GABA, glycine, taurine, and phosphoethanolamine increased significantly within 50–80 min after the infusion of 10 mg of protamine sulfate. CSF albumin levels were significantly increased 1 h after infusion. We conclude that a dysfunction of the BBB, of a degree known to induce brain edema (10 mg of protamine sulfate), significantly increases the extracellular concentration of excitatory amino acids, GABA, taurine, and phosphoethanolamine in the extracellular space.     

Availability of assimilates and formation of aroma compounds in apples as affected by the fruit/leaf ratio

Availability of assimilates in apple trees ( Malus domestica cv. Jonagored) was affected by removing young fruits to obtain 3 ranges of fruit/leaf ratios with average values of 130, 268 and 381 fruits per kg leaf dry matter. Fruit analyses were carried out at fruit harvest and 4 times during a 3-week ripening period. The analyses included detection of volatile aroma components from the juice by headspace gas chromatography. At a low fruit/leaf ratio, higher concentrations of total dry matter, soluble solid and titrateable acids were found. The flesh was also firmer, and ethylene development proceeded at a lower rate and reached a lower maximum value. Aroma compounds consisted of ca 20% esters, 73% alcohols and 6% C-6 aldehydes. The production of butylacetate and hexylacetate, which were the dominating esters, peaked during the ripening period and was most pronounced at the lowest fruit/leaf ratios. At the last sampling date this was also the case for butanol, which was the dominating alcohol. Other esters and alcohols behaved similarly, while C-6 aldehydes showed no significant differences in the fruit/leaf ratio. We suggest that the greater availability of assimilates when internal competition is relieved at a low fruit/leaf ratio causes increased accumulation of fatty acid aroma precursors and aroma compounds as well as of sugars, acids and other compounds in the fruits.     

On the Development of the Eminentia Mediana of the Hypophysis in Rana temporaria, Studied in Normal, Hypophysectomized, and Thyroidectomized Tadpoles.

In the Rana temporaria tadpole, the part of the infundibular floor that eventually becomes the eminentia mediana is very thin throughout premetamorphosis; the supply of aminergic and neurosecretory nerves is poor, and the area is covered by a few fenestrated capillaries. The organ begins to thicken around mid-prometamorphosis owing to a marked invasion of nerves. At this stage it is also penetrated by capillaries and perivascular space extensions, which together form the neurovascular link structures. These become more pronounced during metamorphic climax, when the eminentia mediana attains the adult appearance. Development of the organ is arrested at the premetamorphic stage after thyroidectomy in the young premetamorphic tadpole. Removal of the adenohypophysial primordium from the embryo of the tailbud stage prevents the vascular—but not the neural—component of the eminentia from developing beyond the premetamorphic stage. By contrast, in the American Rana -species investigated the neural component also fails to develop after hypophysectomy.     

Primary and secondary seed dispersal by wind and water in Spergularia salina

The patterns of repositioning by wind and water following their initial dispersal from the parent plant, of winged and unwinged seeds of the heteromorphic halophyte Spergularia salina were Investigated experimentally in both dense vegetation and bare ground under field conditions in a sea shore meadow in eastern Sweden Seeds were placed in situ in the field, and after four days with wind as the sole dispersing agency, 19% of the seeds were repositioned After another 11 days, during which both wind and water acted as dispersing agencies, all seeds of both types had either become repositioned and were still visible (1/3 of the seeds), had penetrated into the ground at the point of release or after dispersal (1/3), or were not recovered (1/3) The probability to become lifted secondarily by water was similar in both seed types Of those seeds repositioned and recovered on the ground, more of the winged type had been transported any distance horizontally than the unwinged type The seed dispersal curve was strongly skewed to the left, and the winged seed type was transported slightly further than the unwinged type, both during primary and secondary dispersal All seeds were transported further when placed on bare soil than when placed in dense vegetation Vertical transportation was quicker in dense vegetation, and unwinged seeds disappeared more quickly into the ground In dense vegetation, unwinged seeds were more frequently encountered in the seed bank than winged seeds, whereas in the absence of vegetation cover, seeds of both types recovered in the soil were found in equal shares     

Secondary chemistry and ribosomal DNA data congruencies in Arnica (Asteraceae)

To investigate possible congruencies between DNA sequence data and secondary chemistry, we compared nuclear ribosomal DNA (nrDNA) sequence data, sesquiterpene lactone (STL) contents, and cytometric data from 35 accessions of 16 Arnica (Asteraceae) species and two outgroup taxa ( Layia hieracioides and Madia sativa ), using phylogenetic inference and principal component analysis (PCA). Several groups supporting multiple accessions of the same species (of A. montana , A. longifolia , A. gracilis , and A. chamissonis ) are congruent between the phylogenetic trees based on nrDNA and STL data. Sesquiterpene lactone profiles were found to be highly consistent within multiple samples of A. montana and A. longifolia respectively . Moreover, sesquiterpene lactone data support subspecies classifications of A. chamissonis and A. parryi , with additional support from DNA sequence data and cytometric data. Morphology, STL data (PCA), cytometric data and DNA sequence data suggest a hybrid origin of one accession ( A. gracilis  ×  longifolia ). In A. gracilis , A. latifolia , and Layia hieracioides , previously not investigated for STLs, we found large amounts of xanthalongin derivatives. This is the first time STLs have been reported from subtribe Madiinae.
© The Willi Hennig Society 2009.