Transport of bacterial inoculants through intact cores of two different soils as affected by water percolation and the presence of wheat plants

Abstract Water flow-innduced transport of Burkholderia cepacia strain P2 and Pseudomonas fluorescens strain R2f cells through intact cores of loamy sand and silt loam field soils was measured for two percolation regimes, 0.9 and 4.4 mm h⁻¹, applied daily during 1 hour. For each strain, transport was generally similar between the two water regimes. Translocation of B. cepacia , with 4.4 mm h⁻¹, did occur initially in both soils. In the loamy sand soil, no change in the bacterial distribution occurred during the experiment (51 days). In the silt loam, B. cepacia cell numbers in the lower soil layers were significantly reduced, to levels at or below the limit of detection. Transport of P. fluorescens in both soils also occurred initially and was comparable to that of B. cepacia . Later in the experiment, P. fluorescens was not detectable in the lower soil layers of the loamy sand cores, due to a large decrease in surviving cell numbers. In the silt loam, the inoculant cell distribution did not change with time. Pre-incubation of the inoculated cores before starting percolation reduced B. cepacia inoculant transport in the loamy sand soil measured after 5 days, but not that determined after 54 days. Delayed percolation in the silt loam soil affected bacterial transport only after 54 days. The presence of growing wheat plants overall enhanced bacterial translocation as compared to that in unplanted soil cores, but only with percolating water. Percolation water from silt loam cores appeared the day after the onset of percolation and often contained inoculant bacteria. With loamy sand, percolation water appeared only 5 days after the start of percolation, and no inoculant bacteria were found. The results presented aid in predicting the fate of genetically manipulated bacteria in a field experiment.     

Reestablishment of self tolerance by suppressor T-cells after active Heymann's nephritis

Adoptive transfer experiments were performed to obtain evidence that the down-regulation of the autoimmune response in rats with active Heymann's nephritis (HN) is due to suppressor T cells. Late in the course of HN antigen-specific OX8+ suppressor T cells were found in the spleen, but never in the draining lymph nodes. These cells were shown to suppress the autoimmune response when transferred to naive recipients that were subsequently challenged. By mixing B cells or helper T cells from rats with HN with suppressor T cells from high-dose tolerant rats we showed that OX8+ suppressor T cells appeared to have a direct suppressive effect on autoreactive B cells. A profound suppressive effect on helper T cells appeared after 10 weeks. Possible mechanisms to account for the failure of Lewis rats to maintain self tolerance are discussed.     

Exonuclease V from Saccharomyces cerevisiae. A 5'----3'-deoxyribonuclease that produces dinucleotides in a sequential fashion

A novel deoxyribonuclease, exonuclease V, has been purified approximately 30,000-fold from Saccharomyces cerevisiae. Exonuclease V is localized in the nucleus. The nuclease degrades single-stranded, but not double-stranded, DNA from the 5'-end. The products of exonuclease action are dinucleotides, except the 3'-terminal tri- and tetranucleotides which are not degraded. Studies with synthetic oligo- and polynucleotides with specified sequence elements showed that exonuclease V cleaves off dinucleotides as primary digestion products. Thus, the polymers (pT)9pA(pT)n and (pT)10pA(pT)n yielded pTpA and pApT as digestion products, respectively. Removal of the 5'-terminal phosphate from the DNA substrate results in reduced binding of the enzyme to the substrate. In addition, the initial hydrolytic cut by exonuclease V on the dephosphorylated substrate produces a mixture of dinucleoside monophosphates and trinucleoside diphosphates. The enzyme is processive in action.     

Two modes of FEN1 binding to PCNA regulated by DNA

The FEN1 nuclease functions during Okazaki fragment maturation in the eukaryotic cell. Like many other proliferating cell nuclear antigen (PCNA)-binding proteins, FEN1 interacts with the interdomain connector loop (IDCL) of PCNA, and PCNA greatly stimulates FEN1 activity. A yeast IDCL mutant pcna-79 (IL126,128AA) failed to interact with FEN-1, but, surprisingly, pcna-79 was still very active in stimulating FEN1 activity. In contrast, a C-terminal mutant pcna-90 (PK252,253AA) showed wild-type binding to FEN1 in solution, but poorly stimulated FEN1 activity. When PCNA was loaded onto a DNA substrate coupled to magnetic beads, it stabilized retention of FEN1 on the DNA. In this DNA-dependent binding assay, pcna-79 also stabilized retention of FEN1, but pcna-90 was inactive. Therefore, in the absence of DNA, FEN1 interacts with PCNA mainly through the IDCL. However, when PCNA encircles the DNA, the C-terminal domain of PCNA rather than its IDCL is important for binding FEN1. An FF-->GA mutation in the PCNA-interaction domain of FEN1 severely decreased both modes of interaction with PCNA and resulted in replication and repair defects in vivo.     

Saccharomyces cerevisiae replication factor C. II. Formation and activity of complexes with the proliferating cell nuclear antigen and with DNA polymerases delta and epsilon.

Lag times in DNA synthesis by DNA polymerase delta holoenzyme were due to ATP-mediated formation of an initiation complex on the primed DNA by the polymerase with the proliferating cell nuclear antigen (PCNA) and replication factor C (RF-C). Lag time analysis showed that high affinity binding of RF-C to the primer terminus required PCNA and that this complex was recognized by the polymerase. The formation of stable complexes was investigated through their isolation by Bio-Gel A-5m filtration. A stable complex of RF-C and PCNA on primed single-stranded mp18 DNA was isolated when these factors were preincubated with the DNA and with ATP, or, less efficiently with ATP gamma S. These and additional experiments suggest that ATP binding promotes the formation of a labile complex of RF-C with PCNA at the primer terminus, whereas its hydrolysis is required to form a stable complex. Subsequently, DNA polymerase delta binds to either complex in a replication competent fashion without further energy requirement. DNA polymerase epsilon did not associate stably with RF-C and PCNA onto the DNA, but its transient participation with these cofactors into a holoenzyme-like initiation complex was inferred from its kinetic properties and replication product analysis. The kinetics of the elongation phase at 30 degrees, 110 nucleotides/s by DNA polymerase delta holoenzyme and 50 nucleotides/s by DNA polymerase epsilon holoenzyme, are in agreement with in vivo rates of replication fork movement in yeast. A model for the eukaryotic replication fork involving both DNA polymerase delta and epsilon is proposed.     

鼠李糖乳杆菌LV108及其发酵乳免疫调节作用的比较

目的 探讨鼠李糖乳杆菌LV108及其发酵乳对免疫抑制小鼠免疫功能的调节作用。 方法 将BALB/c小鼠随机分为5组,每组10只,即空白组(正常小鼠)、模型组(免疫抑制小鼠)、药物组(免疫抑制小鼠食物中添加左旋咪唑)、LV108菌悬液组(免疫抑制小鼠食物中添加LV108菌悬液)和LV108发酵乳组(免疫抑制小鼠食物中添加LV108发酵乳),除空白组外其余组构建免疫抑制小鼠模型。干预4周后,分别测定各组小鼠体质量和脏器指数,血清中白细胞介素2(IL2)、肿瘤坏死因子α(TNFα)和免疫球蛋白G(IgG)含量,血清溶血素含量、耳肿胀度和肝、脾巨噬细胞吞噬能力。 结果 相比模型组,LV108菌悬液组和LV108发酵乳组小鼠体质量增长速度、脏器指数、血清IL2与IgG水平、血清溶血值、耳肿胀度和巨噬细胞吞噬能力显著升高(均P结论 LV108菌体及发酵乳对免疫抑制小鼠具备较全面的免疫调节作用,均可提高小鼠的自身免疫力;LV108发酵乳对小鼠的免疫调节作用强于LV108菌体。     

Pif1 helicase directs eukaryotic Okazaki fragments toward the two-nuclease cleavage pathway for primer removal

Eukaryotic Okazaki fragment maturation requires complete removal of the initiating RNA primer before ligation occurs. Polymerase delta (Pol delta) extends the upstream Okazaki fragment and displaces the 5'-end of the downstream primer into a single nucleotide flap, which is removed by FEN1 nuclease cleavage. This process is repeated until all RNA is removed. However, a small fraction of flaps escapes cleavage and grows long enough to be coated with RPA and requires the consecutive action of the Dna2 and FEN1 nucleases for processing. Here we tested whether RPA inhibits FEN1 cleavage of long flaps as proposed. Surprisingly, we determined that RPA binding to long flaps made dynamically by polymerase delta only slightly inhibited FEN1 cleavage, apparently obviating the need for Dna2. Therefore, we asked whether other relevant proteins promote long flap cleavage via the Dna2 pathway. The Pif1 helicase, implicated in Okazaki maturation from genetic studies, improved flap displacement and increased RPA inhibition of long flap cleavage by FEN1. These results suggest that Pif1 accelerates long flap growth, allowing RPA to bind before FEN1 can act, thereby inhibiting FEN1 cleavage. Therefore, Pif1 directs long flaps toward the two-nuclease pathway, requiring Dna2 cleavage for primer removal.     

Uracil recognition by replicative DNA polymerases is limited to the archaea, not occurring with bacteria and eukarya

Family B DNA polymerases from archaea such as Pyrococcus furiosus, which live at temperatures ~100°C, specifically recognize uracil in DNA templates and stall replication in response to this base. Here it is demonstrated that interaction with uracil is not restricted to hyperthermophilic archaea and that the polymerase from mesophilic Methanosarcina acetivorans shows identical behaviour. The family B DNA polymerases replicate the genomes of archaea, one of the three fundamental domains of life. This publication further shows that the DNA replicating polymerases from the other two domains, bacteria (polymerase III) and eukaryotes (polymerases δ and ε for nuclear DNA and polymerase γ for mitochondrial) are also unable to recognize uracil. Uracil occurs in DNA as a result of deamination of cytosine, either in G:C base-pairs or, more rapidly, in single stranded regions produced, for example, during replication. The resulting G:U mis-pairs/single stranded uracils are promutagenic and, unless repaired, give rise to G:C to A:T transitions in 50% of the progeny. The confinement of uracil recognition to polymerases of the archaeal domain is discussed in terms of the DNA repair pathways necessary for the elimination of uracil.     

Identifying Ca2+-Binding Sites in Proteins by Liquid Chromatography-Mass Spectrometry Using Ca2+-Directed Dissociations

Here we describe a new method to identify calcium-binding sites in proteins using high-resolution liquid chromatography-mass spectrometry in concert with calcium-directed collision-induced dissociations. Our method does not require any modifications to the liquid chromatography-mass spectrometry apparatus, uses standard digestion protocols, and can be applied to existing high-resolution MS data files. In contrast to NMR, our method is applicable to very small amounts of complex protein mixtures (femtomole level). Calcium-bound peptides can be identified using three criteria: (1) the calculated exact mass of the calcium containing peptide; (2) specific dissociations of the calcium-containing peptide from threonine and serine residues; and (3) the very similar retention times of the calcium-containing peptide and the free peptide.Calcium-dependent protein interactions mostly organized in protein networks are responsible for the regulation of cell cycle progression, cell growth, differentiation, secretion, and cytoskeletal organization (1–3). As many of these proteins are linked to various pathological conditions, they are clinically important. The speed at which calcium can have an interplay between various cellular components is impressive and comes notably detectable in neurological processes and in muscle contraction. Calcium binding sites in proteins can be determined by NMR spectroscopy (4, 5). For example, by such NMR measurements, the Ca²⁺-binding sites of the tellurite-resistance protein TerD from Klebsiella pneumoniae were found to be formed in part by a highly conserved motif of 13 residues specified by the sequence GDN(R/L)TG(E/A)GDGDDE (4).Although NMR is the gold standard to study calcium binding in proteins, this approach has several drawbacks. For instance, protein size is limited (< 30 kDa) and proteins should be pure and isotopically labeled. In addition, although the information content is high, NMR is relatively insensitive compared with other techniques such as MS and fluorescence spectroscopy, and relatively large quantities of material (typically 0.5 ml at 0.5–1.0 mm in biological samples) are needed, although efforts are devoted to improve sensitivity in NMR, such as stripline NMR (6).In bottom-up proteomics, proteolytic peptides, generated by enzymatic digestion of complex protein mixtures, are sequenced by MS-based methods (MS/MS (7, 8)) using collision-induced dissociations. Because of the even higher complexity of these peptide mixtures, liquid chromatography (LC)¹ is used to separate the peptides prior to sequencing. In such an LC-MS/MS procedure, many peptides can be identified belonging to the same protein. It has been stated (9) that by this procedure more peptides are analyzed than strictly necessary for identification purposes, but it can equally well be argued that such large coverages enable more reliable protein identifications; moreover, these larger coverages allow the detection of post-translational modifications, including specific calcium complexation as described here.Considering the need of identifying calcium-bound proteins in complex biological samples at low concentrations, we set out to develop a novel method for detecting Ca²⁺-binding sites in proteins based on LC-MS.     

An antigen resembling HL-A7 on the leukocytes of vervet monkeys.

Human leukocyte typing sera of known specificities were used to test the leukocyte antigens of vervet monkeys. The results suggest that these leukocytes contained an antigen resembling the HL-A7 antigen of human leukocytes. This is similar to a previous observation with leukocytes from baboons. These findings are consistent with the suggestion that the 4a/4b complex is the precursor substance from which the other specificities have evolved.