First Experimental Characterization of Microwave Emission from Cosmic Ray Air Showers.

We report the first direct measurement of the overall characteristics of microwave radio emission from extensive air showers. Using a trigger provided by the KASCADE-Grande air shower array, the signals of the microwave antennas of the Cosmic-Ray Observation via Microwave Emission experiment have been read out and searched for signatures of radio emission by high-energy air showers in the GHz frequency range. Microwave signals have been detected for more than 30 showers with energies above 3×10^{16} eV. The observations presented in this Letter are consistent with a mainly forward-directed and polarized emission process in the GHz frequency range. The measurements show that microwave radiation offers a new means of studying air showers at E≥10^{17} eV.

Co-cultivation of a bacteriocin-producing mixed culture of Bifidobacterium thermophilum RBL67 and Pediococcus acidilactici UVA1 isolated from baby faeces.

AIMS: We investigated the kinetics of growth, metabolism and antimicrobial activity of Bifidobacterium thermophilum RBL67 and Pediococcus acidilactici UVA1 previously isolated as a consortium from human baby faeces and producing antimicrobial proteinaceous compounds. METHODS AND RESULTS: Cell growth, antimicrobial activity, glucose consumption, organic acid production and pediocin-gene expression were monitored during pure and mixed strain batch cultures with controlled pH (6.0) at 37 degrees C. The balance of the two strains in mixed cultures was stable, yielding high cell count of 10(9) CFU ml(-1) after a very short incubation time of 4 h for UVA1 and 7 h for RBL67, and RBL67 was not affected by the high production of pediocin by UVA1 during co-culture (up to 12.8 microg ml(-1)). Furthermore, a real-time PCR assay was developed and allowed gene-expression analysis of the pediocin-gene pedA. CONCLUSIONS: The co-culture of RBL67 and UVA1 showed high stability, cell yields and bacteriocin production during batch cultures and has potential for use as a probiotic mixture with antibacterial properties. In addition, a gene-expression real-time PCR assay was successfully developed, used for the relative quantification of the pediocin transcript pedA and demonstrated to be a valuable complementary tool to activity assay. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study reporting of a stable mixed culture of two bacteriocin-producing strains of human origin.

Genetic evidence of an essential role for cytomegalovirus small capsid protein in viral growth.

Many steps in the replication cycle of cytomegalovirus (CMV), like cell entry, capsid assembly, and egress of newly synthesized virions, have not been completely analyzed yet. In order to facilitate these studies, we decided to construct a recombinant CMV that incorporates the green fluorescent protein (GFP) into the nucleocapsid. A comparable herpes simplex virus type 1 (HSV-1) mutant has recently been generated by fusion of the GFP open reading frame (ORF) with the HSV-1 ORF encoding small capsid protein (SCP) VP26 (P. Desai and S. Person, J. Virol. 72:7563-7568, 1998). Recombinant CMV genomes expressing a fusion protein consisting of GFP and the SCP were constructed by the recently established bacterial artificial chromosome mutagenesis procedure. In transfected cells, the SCP-GFP fusion protein localized to distinct foci in the nucleus that may represent sites for capsid assembly (assemblons). However, no viable progeny was reconstituted from these mutant CMV genomes. CMV genomes with deletion of the SCP ORF also did not give rise to infectious virus. Rescue of the mutation by insertion of the SCP gene at an ectopic position in an SCP knockout genome indicates that, in contrast to the HSV-1 SCP, the CMV SCP is essential for viral growth. Expression of the SCP-GFP fusion protein together with the authentic SCP blocked the CMV infection cycle, suggesting that the SCP-GFP fusion protein exerts a dominant-negative effect on the assembly of new virions. The results of this study are discussed with regard to recently published data about the structure of the CMV virion and its differences from the HSV-1 virion.

Characterization of a self-splicing mini-intein and its conversion into autocatalytic N- and C-terminal cleavage elements: facile production of protein building blocks for protein ligation.

The determinants governing the self-catalyzed splicing and cleavage events by a mini-intein of 154 amino acids, derived from the dnaB gene of Synechocystis sp. were investigated. The residues at the splice junctions have a profound effect on splicing and peptide bond cleavage at either the N- or C-terminus of the intein. Mutation of the native Gly residue preceding the intein blocked splicing and cleavage at the N-terminal splice junction, while substitution of the intein C-terminal Asn154 resulted in the modulation of N-terminal cleavage activity. Controlled cleavage at the C-terminal splice junction involving cyclization of Asn154 was achieved by substitution of the intein N-terminal cysteine residue with alanine and mutation of the native C-extein residues. The C-terminal cleavage reaction was found to be pH-dependent, with an optimum between pH6.0 and 7.5. These findings allowed the development of single junction cleavage vectors for the facile production of proteins as well as protein building blocks with complementary reactive groups. A protein sequence was fused to either the N-terminus or C-terminus of the intein, which was fused to a chitin binding domain. The N-terminal cleavage reaction was induced by 2-mercaptoethanesulfonic acid and released the 43kDa maltose binding protein with an active C-terminal thioester. The 58kDa T4 DNA ligase possessing an N-terminal cysteine was generated by a C-terminal cleavage reaction induced by pH and temperature shifts. The intein-generated proteins were joined together through a native peptide bond. This intein-mediated protein ligation approach opens up novel routes in protein engineering.

[Optimizing plasma cell content in bone marrow aspirates]. / Optimierung der Ausbeute von Plasmazellen in Knochenmarkaspiraten.

Bone marrow samples used for flow cytometric analysis of plasma cells frequently provide a low plasma cell content. Regularly, samples used for flow cytometry are provided by second step aspiration while the first aspirate is used for cytologic examination. We investigated whether the use of secondary aspirates leads to a systematic underestimation of the bone marrow plasma cell content as a consequence of an increased blood contaminant. To test the hypothesis, plasma cell (CD38bright) percentages were established by flow cytometry in 13 pairs of primary/secondary aspirates. In all cases we found lower plasma cell contents in secondary as compared to primary aspirates (p = 0.0015). Median plasma cell counts in secondary aspirates were 57% lower compared to primary aspirates. We conclude that the use of secondary aspirates leads to systematic underestimation of the bone marrow plasma cell content.

Evaluation of the Sysmex R-1000. An automated reticulocyte analyzer.

The new fully automated reticulocyte analyzer, Sysmex R-1000 (TOA Medical Electronics, Kobe, Japan), was evaluated for its routine use in the Hematological Laboratory at the University Hospital Basel, Switzerland. The operating characteristics, such as within-run precision, linearity, and carryover, fulfilled the manufacturer's specifications and are excellent. Correlation with the standard method, manual reticulocyte counting, is linear for normal and high values. For low reticulocyte counts the regression points show a deviation from their linearity. An absolute zero value is not obtained by the R-1000. The R-1000 measures total RNA content of each cell and expresses the value as low fluorescence ratio (LFR), medium fluorescence ratio (MFR), and high fluorescence ratio (HFR). The analysis of this ratio resolves the problem of zero reticulocytes: A fraction of less than 0.002 (0.2%) with an LFR of 100% represents aplasia; a shift of the intensity of fluorescence to HFR heralds regeneration. Results of samples stored at room temperature remain stable and within the range of the within-run precision for up to 12 hours, when stored at 5 degrees C for more than 48 hours. The authors conclude that the R-1000 is easy to operate, fulfills the criteria for accuracy and precision, and is highly suitable for daily routine use in a large central hematologic laboratory.