A new approach for rhizosphere research by X-ray microanalysis of microliter soil solutions

Lolium perenne growing with high root density on a fine nylon mesh (Kuchenbuch and Jungk, 1982) caused the development of element gradients in the rhizosphere below the mesh. Micro-liter soil solutions from 2-mg soil samples were sprayed onto Formvar-coated grids and analyzed by X-ray microanalysis in a transmission electron microscope. The results were comparable to those obtained by flame photometry and atomic absorption spectrometry (AAS) of conventional soil solutions from 1 g soil. X-ray microanalysis of micro-soil solutions allows the application of different extraction procedures to even small amounts of soil usually available from rhizosphere experiments. Information about soil buffering characteristics in the rhizosphere can thus be obtained. Aluminum accumulation in the rhizosphere of small segments of single Picea abies fine roots grown in undisturbed natural forest soil could be detected with this technique.     

Ammonium sensing in nitrogen fixing bacteria: Functions of theglnB andglnD gene products

A plentiful supply of fixed nitrogen as ammonium (or other compounds such as nitrate or amino acids) inhibits nitrogen fixation in free-living bacteria by preventing nitrogenase synthesis and/or activity. Ammonium and nitrate have variable effects on the ability ofRhizobiaceae (Rhizobium, Bradyrhizobium andAzorhizobium) species to nodulate legume hosts and on nitrogen fixation capacity in bacteroid cells contained in nodules or in plant-free bacterial cultures. In addition to effects on nitrogen fixation, excess ammonium can inhibit activity or expression of other pathways for utilization of nitrogenous compounds such as nitrate (through nitrate and nitrite reductase), or glutamine synthetase (GS) for assimilation of ammonium. This paper describes the roles of two key genesglnB andglnD, whose gene products sense levels of fixed nitrogen and initiate a cascade of reactions in response to nitrogen status. While work onEscherichia coli and other enteric bacteria provides the model system,glnB and, to a lesser extent,glnD have been studied in several nitrogen fixing bacteria. Such reports will be reviewed here. Recent results on the identity and function of theglnB andglnD gene products inAzotobacter vinelandii (a free-living soil diazotroph) and inRhizobium leguminosarum biovarviciae, hereinafter designatedR.l. viciae will be presented. New data suggests thatAzotobacter vinelandii probably contains aglnB-like gene and this organism may have twoglnD-like genes (one of which was recently identified and namednfrX). In addition, evidence for uridylylation of theglnB gene product (the PII protein) ofR. l. viciae in response to fixed nitrogen deficiency is presented. Also, aglnB mutant ofR. l. viciae has been isolated; its characteristics with respect to expression of nitrogen regulated genes is described.     

Generation and effective enrichment of selectable human cytomegalovirus mutants using site-directed insertion of the neo gene

Studies on the biology and function of human cytomegalovirus (HCMV) genes have been hampered by the limited number of viral mutants available for genetic analyses. We have developed a simple procedure to generate and enrich for HCMV recombinants. By inserting the bacterial neo gene, encoding neomycin/kanamycin phosphotransferase, into the large HCMV DNA genome using homologous recombination, selectable mutants of this complex herpesvirus were isolated for the first time. The synthesis of Neo from the viral genome was used to effectively enrich for recombinant viruses (re-viruses) in permissive culture cells grown in the presence of Geneticin (G418). A quick assay for Neo activity in infected cells, based on phosphorylation of kanamycin (Km), was used to easily identify viral recombinants in the process of screening and isolation. This procedure, not used previously to identify re-viruses, proved to be very useful for screening of large numbers of HCMV recombinants. Analysis of re-virus by Southern blotting revealed that the insertion of the marker gene had resulted in the expected deletion of the open reading frames, TRL 13/14 and UL 1–5, of HCMV. Re-virus was stable and showed no differences in growth kinetics as compared to wild-type (wt) virus. The insertion of a selectable marker gene into the HCMV genome and identification of viral recombinants by the Km phosphorylation assay, as presented here, provides the rationale for effective generation, enrichment and stable propagation of HCMV mutants.     

Crystalline bacterial cell surface layers

Crystalline arrays of proteinaceous subunits forming surface layers (S-layers) are one of the most commonly observed prokaryotic cell envelope structures. They are ubiquitous amongst Gram-positive and Gram-negative archaeobacteria and eubacteria and, if present, account for the major protein species produced by the cells. S-layers can provide organisms with a selection advantage by providing various functions including protective coats, molecular sieves, ion traps and structures involved in cell surface interactions. S-layers were identified as contributing to virulence when present as a structural component of pathogens. In Gram-negative archaeobacteria they are involved in determining cell shape and cell division. The crystalline arrays reveal a broad-application potential in biotechnology, vaccine development and molecular nanotechnology.     

L cells expressing DQ molecules of the DR3 and DR4 haplotypes: reactivity patterns with mAbs

cDNAs coding for the HLA class II DR and DQ and chains of the diabetogenic haplotypes DR3 and DR4 were introduced into a mammalian expression vector and transfected into L-cell mouse fibroblasts to produce cells expressing individual human class II molecules. Stable L transfectants were generated expressing each of the DR or DQ isotypes of the cis-encoded and chains of the DR3 or DR4 haplotypes, as well as the trans-encoded and chains of the DQ molecules of the two haplotypes. However, isotype mismatched combinations (DR/DQ or DQ/DR) did not result in any stable transfectants. The stable DQ L-cell transfectants obtained, along with homozygous B-cell lines expressing the DQ2 and DQ8 specificities, were tested against a large panel of twentyone anti-HLA class II monoclonal antibodies (mAbs). Their unusual reactivity patterns are described including the failure of most pan-DQ mAbs to react with all DQ expressing L-cell transfectants. Interestingly, some mAbs react with certain heterodimers expressed on B-LCL but fail to recognize the same heterodimers expressed on the transfectants. This is suggestive of minor structural modifications that class II molecules undergo depending on the cells they are expressed on.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number U07848. The name DQB1 ^* 0202 was officially assigned by the WHO Nomenclature Committee in April 1994. This follows the agreed policy that, subject to the conditions stated in the most recent Nomenclature Report (Bodmer et al. 1992), names will be assigned to new sequences as they are identified. Lists of such new names will be published in the following WHO Nomenclature Report     

NMDA Receptor Heterogeneity During Postnatal Development of the Rat Brain: Differential Expression of the NR2A, NR2B, and NR2C Subunit Proteins

Abstract: Changes in the expression of the NMDA receptor subunits (NRs) NR2A, 2B, and 2C were investigated in histo blots of the developing rat brain with subunit-specific antisera. At birth, the NR2B subunit was detected almost ubiquitously, the NR2A subunit staining was faint and restricted to the hippocampus, cerebral cortex, and striatum, and no NR2C subunit immunoreactivity was detected. During the first 3 postnatal weeks, the NR2B subunit became confined to forebrain structures, whereas the NR2A immunoreactivity became abundantly expressed throughout the brain. The NR2C immunoreactivity emerged 5 days after birth in the olfactory bulb, thalamus, and vestibular nuclei and became very intense after 10 days in cerebellar granule cells, its primary site of expression in adulthood. After 3 weeks, NR2A and NR2B immunoreactivity decreased to adult levels, whereas NR2C immunoreactivity remained unchanged. The patterns of distribution of the subunit proteins were in agreement with those of their corresponding mRNAs, as monitored by in situ hybridization histochemistry, although the mRNA translation appeared to be delayed by several days in certain areas. Our results reveal a progressive increase in the heterogeneity of NMDA receptors due to the comparably late onset of NR2A and NR2C subunit expression and by the area-specific rearrangement of NR2B subunit expression following birth.     

In vitro degradation of guanosine 3′,5′-bis(diphosphate) [ppGpp] by the spoT gene product [ppGppase] from auxotrophic strains of Escherichia coli: Effects of various antibiotics and drugs

The enzyme specifically hydrolyzing guanosine 3,5-bis(diphosphate) [ppGpp] has been isolated from the ribosomal fraction of Escherichia coli; it released pyrophosphate from the 3-position of ppGpp. The effects of various drugs and antibiotics known to interfere with protein and/or RNA synthesis were investigated in the ppGpp degrading reaction. It was determined that tetracycline, chlorotetracycline, and thiostrepton strongly inhibited the reaction, whereas levallorphan gave a moderate inhibition. Only the tetracycline-mediated inhibition could be reversed by manganese ions. Oxytetracycline, rifampicin, fusidic acid, kirromycin, streptomycin, puromycin, chloramphenicol, and morphine did not inhibit the decay reaction.Abbreviations ppGpp guanosine 3,5-bis(diphosphate)     

Lipid and Phenolic Constituents from Seeds of Hypericum perforatum L. and Hypericum tetrapterum Fr. and their Antioxidant Activity

Seeds of Hypericum perforatum and H. tetrapterum were extracted with dichloromethane and methanol and investigated by chromatographic and mass spectrometric methods. Both species yielded a fatty oil fraction amounting to 30.5% and 18.0% of the seed weight, respectively. Linoleic acid (C18:2n‐6) was shown to be the predominant fatty acid constituent. Moreover, xanthone derivatives, i.e. tetrahydroxyxanthones (THX), xanthone‐glycosides and xanthone‐sulfonates, were assigned in methanolic extracts. For structure elucidation, one representative xanthone, namely 1,3,6,7‐THX, was synthesized and analyzed via HPLC‐DAD/MSⁿ and GC/MS. Total THX contents were quantitated applying a validated HPLC‐DAD method, resulting in 1.25 g/kg (H. perforatum) and 0.27 g/kg (H. tetrapterum), respectively. Moreover, the free radical scavenging capacity of the methanol extracts was tested using the DPPH antioxidant assay. Both, H. perforatum (IC₅₀ = 8.7 mg/l) and 1,3,6,7‐THX (IC₅₀ = 3.0 mg/l), exhibited good DPPH free radical scavenging activity compared to Trolox (IC₅₀ = 6.6 mg/l).     

Taxonomic aggregation does not alleviate the lack of consistency in analysing diversity in long‐term phytoplankton monitoring data: a rejoinder to Pomati et al. (2015)

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