Predicted class-I aminoacyl tRNA synthetase-like proteins in non-ribosomal peptide synthesis

Background  

Recent studies point to a great diversity of non-ribosomal peptide synthesis systems with major roles in amino acid and co-factor biosynthesis, secondary metabolism, and post-translational modifications of proteins by peptide tags. The least studied of these systems are those utilizing tRNAs or aminoacyl-tRNA synthetases (AAtRS) in non-ribosomal peptide ligation.     

Reconstitution of RecBC DNase activity from purified Escherichia coli RecB and RecC proteins

The Escherichia coli RecB protein, normally synthesized in low amounts, has been amplified by linkage of the recB gene to the phage lambda leftward promoter in an expression plasmid. From strains harboring this plasmid, RecB protein has been purified to homogeneity by a simple procedure which includes affinity chromatography on a column of RecC protein bound to agarose. The purified RecB protein has DNA-dependent ATPase activity but no exonuclease activity. RecC protein alone has neither ATPase nor exonuclease activity. However, when combined together, the RecB and RecC proteins show the ATP-dependent double-stranded exonuclease properties characteristic of the RecBC DNase.     

Choline transport in Fusarium graminearum A 3 5

Fusarium graminearum A 3/5 possesses a high affinity system (Km = 32 +/- 8 microM; mean +/- SE) for uptake of choline, which was shown to be energy-dependent and constitutive. The maximum rate of choline uptake by this system was repressed by ammonia and glucose, showing a three-fold increase in maximum activity after nitrogen (2 h) or carbon (4 h) starvation. The system was highly specific for choline with only dimethylethanolamine (Ki = 198 +/- 29 microM), betaine aldehyde (Ki = 95 +/- 14 microM) and chlorocholine (Ki = 352 +/- 40 microM) acting as competitive inhibitors. Hemicholinium-3 acted as a mixed (non-competitive) inhibitor (KIES = 1.9 +/- 0.6 microM; KIE = 3.6 +/- 1.9 microM).     

Promoting Smoke-Free Homes: A Novel Behavioral Intervention Using Real-Time Audio-Visual Feedback on Airborne Particle Levels

Interventions are needed to protect the health of children who live with smokers. We pilot-tested a real-time intervention for promoting behavior change in homes that reduces second hand tobacco smoke (SHS) levels. The intervention uses a monitor and feedback system to provide immediate auditory and visual signals triggered at defined thresholds of fine particle concentration. Dynamic graphs of real-time particle levels are also shown on a computer screen. We experimentally evaluated the system, field-tested it in homes with smokers, and conducted focus groups to obtain general opinions. Laboratory tests of the monitor demonstrated SHS sensitivity, stability, precision equivalent to at least 1 µg/m³, and low noise. A linear relationship (R² = 0.98) was observed between the monitor and average SHS mass concentrations up to 150 µg/m³. Focus groups and interviews with intervention participants showed in-home use to be acceptable and feasible. The intervention was evaluated in 3 homes with combined baseline and intervention periods lasting 9 to 15 full days. Two families modified their behavior by opening windows or doors, smoking outdoors, or smoking less. We observed evidence of lower SHS levels in these homes. The remaining household voiced reluctance to changing their smoking activity and did not exhibit lower SHS levels in main smoking areas or clear behavior change; however, family members expressed receptivity to smoking outdoors. This study established the feasibility of the real-time intervention, laying the groundwork for controlled trials with larger sample sizes. Visual and auditory cues may prompt family members to take immediate action to reduce SHS levels. Dynamic graphs of SHS levels may help families make decisions about specific mitigation approaches.     

Prions and lymphoid organs: Solved and remaining mysteries

Prion colonization of secondary lymphoid organs (SLOs) is a critical step preceding neuroinvasion in prion pathogenesis. Follicular dendritic cells (FDCs), which depend on both tumor necrosis factor receptor 1 (TNFR1) and lymphotoxin β receptor (LTβR) signaling for maintenance, are thought to be the primary sites of prion accumulation in SLOs. However, prion titers in RML-infected TNFR1^−/− lymph nodes and rates of neuroinvasion in TNFR1^−/− mice remain high despite the absence of mature FDCs. Recently, we discovered that TNFR1-independent prion accumulation in lymph nodes relies on LTβR signaling. Loss of LTβR signaling in TNFR1^−/− lymph nodes coincided with the de-differentiation of high endothelial venules (HEVs)—the primary sites of lymphocyte entry into lymph nodes. These findings suggest that HEVs are the sites through which prions initially invade lymph nodes from the bloodstream. Identification of HEVs as entry portals for prions clarifies a number of previous observations concerning peripheral prion pathogenesis. However, a number of questions still remain: What is the mechanism by which prions are taken up by HEVs? Which cells are responsible for delivering prions to lymph nodes? Are HEVs the main entry site for prions into lymph nodes or do alternative routes also exist? These questions and others are considered in this article.     

Genetic tagging of tumor cells with retrovirus vectors: clonal analysis of tumor growth and metastasis in vivo.

Retrovirus vector infection was used to introduce large numbers of unique genetic markers into tumor cell populations for the purpose of analyzing comparative changes in the clonal composition of metastatic versus that of nonmetastatic tumors during their progressive growth in vivo. The cell lines used were SP1, a nonmetastatic, aneuploid mouse mammary adenocarcinoma, and SP1HU9L, a metastatic variant of SP1. Cells were infected with delta e delta pMoTN, a replication-defective retrovirus vector which possesses the dominant selectable neo gene and crippled long terminal repeats. G418r colonies were obtained at a frequency of 4 x 10(-3). Southern blot analysis of a number of clones provided evidence of random and heritable integration of one or two copies of the proviral DNA. Clonal evolution of primary tumor growth and the nature of lineage relationships among spontaneous metastases and primary tumors were analyzed by subcutaneously injecting 10(5) cells from a pooled mixture of 3.6 x 10(2) G418r SP1HU9L or 10(4) G418r SP1 colonies into syngeneic CBA/J mice. The most striking finding was the relative clonal homogeneity of advanced primary tumors; they invariably consisted of a small number (less than 10) of distinct clones despite the fact that hundreds or thousands of uniquely marked clones had been injected. In the case of the metastatic SP1HU9L cells, the nature of these "dominant" clones varied from one tumor to another. Analysis of a number of lung metastases revealed that a proportion of them were derived from dominant primary tumor clones and were composed of one, and sometimes two, distinct progenitors. In some animals, all the lung metastases were derived from a common progenitor clone, whereas in others, each metastatic nodule had a different progenitor. The results show the following. (i) Retrovirus vector infection can be used to introduce large numbers of unique and stable clonal markers into tumor cell populations. (ii) The progeny of a very limited number of clones dominate in advanced primary tumors. (iii) Mammary carcinoma metastases are of mono- or biclonal origin. The significance of the results is discussed.