Planococcus rifietensis sp. nov, isolated from algal mat collected from a sulfurous spring in Campania (Italy)

The taxomony of strain M8, isolated from algal mat formed at the origin of a sulfurous spring in Rifieto (Savignano Irpino, Campania, Italy), was investigated in a polyphasic approach. The morphological, physiological and genetic characteristics were compared with of Planococcus and Planomicrobium species. The isolate grew optimally at pH 9.0, 1.8 M NaCl at 37 degrees C. The cells were Gram-positive cocci that form pairs, tetrads and aggregates of several cells. The isolate was aerobic/microaerophilic and accumulated glycine-betaine, as a major osmolyte, with minor components glutamate and an unknown compound. M8 was able to hydrolyse X-Glc (5-bromo-4-chloro-3-indoyl beta-d-glucopyranoside). The polar lipid profile consisted of phosphatidylglycerol and diphosphatidylglycerol as major components, and phosphocholine as a minor compound. MK8 was the only quinone found and the fatty acid composition was dominated by branched acids, mainly aiC15:0. The G+C content of DNA was 47.9% and its phylogenetic position was established by 16S rRNA gene sequencing as a member of the genus Planococcus. The DNA/DNA similarity of M8 to the type species Planococcus citreus was less than 55%. For this reason and for physiological and chemotaxonomic features, it is proposed to create a new species Planococcus rifietensis sp. nov.     

Homo- and hetero-oligomerization of ammonium transporter-1 NH4 uniporters

In most organisms, high affinity ammonium uptake is catalyzed by members of the ammonium transporter family (AMT/MEP/Rh). A single point mutation (G458D) in the cytosolic C terminus of the plasma membrane transporter LeAMT1;1 from tomato leads to loss of function, although mutant and wild type proteins show similar localization when expressed in yeast or plant protoplasts. Co-expression of LeAMT1;1 and mutant in Xenopus oocytes inhibited ammonium transport in a dominant negative manner, suggesting homo-oligomerization. In vivo interaction between LeAMT1;1 proteins was confirmed by the split ubiquitin yeast two-hybrid system. LeAMT1;1 is isolated from root membranes as a high molecular mass oligomer, converted to a approximately 35-kDa polypeptide by denaturation. To investigate interactions with the LeAMT1;2 paralog, co-localizing with LeAMT1;1 in root hairs, LeAMT1;2 was characterized as a lower affinity NH4+ uniporter. Co-expression of wild types with the respective G458D/G465D mutants inhibited ammonium transport in a dominant negative manner, supporting the formation of heteromeric complexes in oocytes. Thus, in yeast, oocytes, and plants, ammonium transporters are able to oligomerize, which may be relevant for regulation of ammonium uptake.     

Adeno-cosmid cloning vectors for regulated gene expression

Background

Adenovectors are widely used for efficient delivery of genes into a variety of cell types and organisms. However, the construction of the desired vector/genes combination, especially if it involves the cloning of several gene cassettes, can be laborious due to the large size of these vectors. New methods are needed to simplify the construction of complex combinations of gene cassettes into adenovectors.

Methods

Using simple cloning techniques and exploiting the λ‐phage packaging system, we devised efficient methods for the ‘selection’ of the desired vector constructs. Thus we generated a series of cosmids containing the adeno helper dependent (HD) backbone in which we inserted cis‐ and trans‐acting tetracycline (tet) elements for the regulation of any gene of interest. One of these cosmids has been used to produce an HD adenovirus carrying a tetracycline‐regulated gene expressing β‐galactosidase.

Results

We have demonstrated that the adeno‐cosmid system allows rapid and efficient cloning of genes of interest in helper dependent vectors, and described a prototype ‘ready‐to‐use’ vector in which any gene of interest can be easily expressed under the control of the tet system. The HD viruses produced with this novel methodology can be grown at high titers, can be easily separated from the helper adenovirus, and allow delivery and regulated gene expression in a variety of tissues.

Conclusions

Exploiting the λ‐packaging system, complex adeno constructs can be generated with a simple and reproducible protocol, which allows selection of the desired size construct, counterselecting for the frequently observed intramolecular recombinations and deletions. Copyright © 2002 John Wiley & Sons, Ltd.

     

Method for antibiotic assay on Sulfolobales

Summary The use of antibiotic-discs on Gelrite-medium, for studying antibiotic sensitivity of Sulfolobales, is reported for the first time.This work was partially supported by an Italian target project PFBBCS.     

Purification of the ADP-ribosylating enzyme from S. solfataricus by SDS-polyacrylamide gel electrophoresis and electroelution

The ADPribosylating enzyme from the thermophilic archaeon S. solfataricus was purified by a simple procedure which included preparative electrophoresis on a 0.1% SDS- polyacrylamide gel. The gel slice containing the enzymatic protein was cut out and the enzyme was solubilized by electroelution. The pure enzyme was obtained by chromatography of the electroeluted sample on a DNA-Sepharose column. The purified enzyme retained both its full activity and the structuring ability as a function of temperature increase.     

Diagnostic Support for Selected Paediatric Pulmonary Diseases Using Answer-Pattern Recognition in Questionnaires Based on Combined Data Mining Applications—A Monocentric Observational Pilot Study

Background

Clinical symptoms in children with pulmonary diseases are frequently non-specific. Rare diseases such as primary ciliary dyskinesia (PCD), cystic fibrosis (CF) or protracted bacterial bronchitis (PBB) can be easily missed at the general practitioner (GP).

Objective

To develop and test a questionnaire-based and data mining-supported tool providing diagnostic support for selected pulmonary diseases.

Methods

First, interviews with parents of affected children were conducted and analysed. These parental observations during the pre-diagnostic time formed the basis for a new questionnaire addressing the parents’ view on the disease. Secondly, parents with a sick child (e.g. PCD, PBB) answered the questionnaire and a data base was set up. Finally, a computer program consisting of eight different classifiers (support vector machine (SVM), artificial neural network (ANN), fuzzy rule-based, random forest, logistic regression, linear discriminant analysis, naive Bayes and nearest neighbour) and an ensemble classifier was developed and trained to categorise any given new questionnaire and suggest a diagnosis. For estimating the diagnostic accuracy, we applied ten-fold stratified cross validation.

Results

All questionnaires of patients suffering from CF, asthma (AS), PCD, acute bronchitis (AB) and the healthy control group were correctly diagnosed by the fusion algorithm. For the pneumonia (PM) group 19/21 (90.5%) and for the PBB group 17/18 (94.4%) correct diagnoses could be reached. The program detected the correct diagnoses with an overall sensitivity of 98.8%. Receiver operating characteristics (ROC) analyses confirmed the accuracy of this diagnostic tool. Case studies highlighted the applicability of the tool in the daily work of a GP.

Conclusion

For children with symptoms of pulmonary diseases a questionnaire-based diagnostic support tool using data mining techniques exhibited good results in arriving at diagnostic suggestions. In the hands of a doctor, this tool could be of value in arousing awareness for rare pulmonary diseases such as PCD or CF.     

Animals as an indicator of carbon sequestration and valuable landscapes

Possibilities of the assessment of a landscape with the use of succession development stages, monitored with the value of the Mean Individual Biomass (MIB) of carabid beetles and the occurrence of bird species are discussed on the basis of an example from Poland. Higher variability of the MIB value in space signifies a greater biodiversity. Apart from the variability of MIB, it is suggested to adopt the occurrence of the following animals as indicators, (in the order of importance), representing underlying valuable landscapes: black stork, lesser spotted eagle, white-tailed eagle, wolf, crane and white stork. The higher number of these species and their greater density indicate a higher value of the landscape for biodiversity and ecosystem services, especially carbon sequestration. All these indicators may be useful to assess measures for sustainable land use.