Cloning and characterization of a ribosomal protein L23a from Haemaphysalis qinghaiensis eggs by immuno screening of a cDNA expression library

A primary cDNA library with a size of 1.34 × 10⁶ PFU was constructed from Haemaphysalis qinghaiensis eggs and was immunoscreened with rabbit anti-H. qinghaiensis serum. One clone (Hq22, named following those clones obtained from adult Haemaphysalis qinghaiensis cDNA library which we constructed before) screened from the cDNA library was selected randomly for sequencing. The entire sequence of the clone was subsequently obtained using rapid amplification of the cDNA ends (RACE). A search of the cloned sequence against GenBank revealed that it related to ribosomal protein L23a (Rpl23a) and had a high percentage similarity to this protein from different species. Conserved domains for Rpl23a were also identified in the cloned sequence. Expression analysis by RT-PCR showed that this gene is expressed in salivary glands, midguts, other tissues and different developmental stages of H. qinghaiensis. Based on the H. qinghaiensis Rpl23a sequence, open reading frames (ORF) of Rpl23a of Heamaphysalis longicornis and Boophilus microplus were also cloned and were performed for comparison with Rpl23a of H. qinghaiensis and other organisms as well. Vaccine based on Rpl23a recombinant protein cannot protect sheep against H. qinghaiensis.     

Filamentous temperature-sensitive Z (FtsZ) isoforms specifically interact in the chloroplasts and in the cytosol of Physcomitrella patens

Plant filamentous temperature-sensitive Z (FtsZ) proteins have been reported to be involved in biological processes related to plastids. However, the precise functions of distinct isoforms are still elusive. Here, the intracellular localization of the FtsZ1-1 isoform in a moss, Physcomitrella patens, was examined. Furthermore, the in vivo interaction behaviour of four distinct FtsZ isoforms was investigated. Localization studies of green fluorescent protein (GFP)-tagged FtsZ1-1 and fluorescence resonance energy transfer (FRET) analyses employing all dual combinations of four FtsZ isoforms were performed in transient protoplast transformation assays. FtsZ1-1 is localized to network structures inside the chloroplasts and exerts influence on plastid division. Interactions between FtsZ isoforms occur in distinct ordered structures in the chloroplasts as well as in the cytosol. The results expand the view of the involvement of Physcomitrella FtsZ proteins in chloroplast and cell division. It is concluded that duplication and diversification of ftsZ genes during plant evolution were the main prerequisites for the successful remodelling and integration of the prokaryotic FtsZ-dependent division mechanism into the cellular machineries of distinct complex processes in plants.     

Jasmonates in arbuscular mycorrhizal interactions

The mutualistic interaction between plants and arbuscular mycorrhizal (AM) fungi is believed to be regulated from the plant side among other signals by the action of phytohormones. Evidences for this are based mainly on application experiments and determination of phytohormone levels in AM roots by comparison to non-mycorrhizal roots. In case of jasmonates, additional proof is given by reverse genetic approaches, which led to first insights into their putative role in the establishment and functioning of the symbiosis. This review summarizes the current data about phytohormone action in AM roots and the role of jasmonates in particular.     

Regulation of arbuscular mycorrhization by carbon. The symbiotic interaction cannot be improved by increased carbon availability accomplished by root-specifically enhanced invertase activity

The mutualistic interaction in arbuscular mycorrhiza (AM) is characterized by an exchange of mineral nutrients and carbon. The major benefit of AM, which is the supply of phosphate to the plant, and the stimulation of mycorrhization by low phosphate fertilization has been well studied. However, less is known about the regulatory function of carbon availability on AM formation. Here the effect of enhanced levels of hexoses in the root, the main form of carbohydrate used by the fungus, on AM formation was analyzed. Modulation of the root carbohydrate status was performed by expressing genes encoding a yeast (Saccharomyces cerevisiae)-derived invertase, which was directed to different subcellular locations. Using tobacco (Nicotiana tabacum) alcc::wINV plants, the yeast invertase was induced in the whole root system or in root parts. Despite increased hexose levels in these roots, we did not detect any effect on the colonization with Glomus intraradices analyzed by assessment of fungal structures and the level of fungus-specific palmitvaccenic acid, indicative for the fungal carbon supply, or the plant phosphate content. Roots of Medicago truncatula, transformed to express genes encoding an apoplast-, cytosol-, or vacuolar-located yeast-derived invertase, had increased hexose-to-sucrose ratios compared to beta-glucuronidase-transformed roots. However, transformations with the invertase genes did not affect mycorrhization. These data suggest the carbohydrate supply in AM cannot be improved by root-specifically increased hexose levels, implying that under normal conditions sufficient carbon is available in mycorrhizal roots. In contrast, tobacco rolC::ppa plants with defective phloem loading and tobacco pyk10::InvInh plants with decreased acid invertase activity in roots exhibited a diminished mycorrhization.     

Single-species microbial biofilm screening for industrial applications

While natural microbial biofilms often consist of multiple species, single-species biofilms are of great interest to biotechnology. The current study evaluates biofilm formation for common industrial and laboratory microorganisms. A total of 68 species of biosafety level one bacteria and yeasts from over 40 different genera and five phyla were screened by growing them in microtiter plates and estimating attached biomass by crystal violet staining. Most organisms showed biofilm formation on surfaces of polystyrene within 24 h. By changing a few simple conditions such as substratum characteristics, inoculum and nutrient availability, 66 strains (97%) demonstrated biofilm formation under at least one of the experimental conditions and over half of these strains were classified as strong biofilm formers, potentially suitable as catalysts in biofilm applications. Many non-motile bacteria were also strong biofilm formers. Biofilm morphologies were visualized for selected strains. A model organism, Zymomonas mobilis, easily established itself as a biofilm on various reactor packing materials, including stainless steel.     

A single 1-h bout of evening exercise increases basal FFA flux without affecting VLDL-triglyceride and VLDL-apolipoprotein B-100 kinetics in untrained lean men

Our group (Magkos F, Wright DC, Patterson BW, Mohammed BS, Mittendorfer B, Am J Physiol Endocrinol Metab 290: E355-E362, 2006) has recently demonstrated that a single, prolonged bout of moderate-intensity cycling (2 h at 60% of peak oxygen consumption) in the evening increases basal whole-body free fatty acid (FFA) flux and fat oxidation, decreases hepatic VLDL-apolipoprotein B-100 (apoB-100) secretion, and enhances removal efficiency of VLDL-triglyceride (TG) from the circulation the following day in untrained, healthy, lean men. In the present study, we investigated the effect of a single, shorter-duration bout of the same exercise (1 h cycling at 60% of peak oxygen consumption) on basal FFA, VLDL-TG, and VLDL-apoB-100 kinetics in seven untrained, healthy, lean men by using stable isotope-labeled tracer techniques. Basal FFA rate of appearance in plasma and plasma FFA concentration were approximately 55% greater (P < 0.05) the morning after exercise than rest, whereas resting metabolic rate and whole-body substrate oxidation rates were not different after rest and exercise. Exercise had no effect on plasma VLDL-TG and VLDL-apoB-100 concentrations, hepatic VLDL-TG and VLDL-apoB-100 secretion rates, and VLDL-TG and VLDL-apoB-100 plasma clearance rates (all P > 0.05). We conclude that in untrained, healthy, lean men 1) the exercise-induced changes in basal whole-body fat oxidation, VLDL-TG, and VLDL-apoB-100 metabolism during the late phase of recovery from exercise are related to the duration of the exercise bout; 2) single sessions of typical recreational activities appear to have little effect on basal, fasting plasma TG homeostasis; and 3) there is a dissociation between systemic FFA availability and VLDL-TG and VLDL-apoB-100 secretion by the liver.     

CD28 deficiency exacerbates joint inflammation upon Borrelia burgdorferi infection, resulting in the development of chronic Lyme arthritis

Lyme disease, caused by the tick-borne spirochete Borrelia burgdorferi (Bb), is a multisystem illness, affecting many organs, such as the heart, the nervous system, and the joints. Months after Bb infection, approximately 60% of patients experience intermittent arthritic attacks, a condition that in some individuals progresses to chronic joint inflammation. Although mice develop acute arthritis in response to Bb infection, the joint inflammation clears after 2 wk, despite continuous infection, only very rarely presenting with chronic Lyme arthritis. Thus, the lack of an animal system has so far prevented the elucidation of this persistent inflammatory process that occurs in humans. In this study, we report that the majority of Bb-infected CD28-/- mice develop chronic Lyme arthritis. Consistent with observations in chronic Lyme arthritis patients, the infected mutant, but not wild-type mice present recurring monoarticular arthritis over an extended time period, as well as anti-outer surface protein A of Bb serum titers. Furthermore, we demonstrate that anti-outer surface protein A Abs develop in these mice only after establishment of chronic Lyme arthritis. Thus, the Bb-infected CD28-/- mice provide a murine model for studying chronic Lyme arthritis.     

TLR signaling mediated by MyD88 is required for a protective innate immune response by neutrophils to Citrobacter rodentium

Enteropathogenic Escherichia coli, enterohemorrhagic E. coli, and Citrobacter rodentium are classified as attaching and effacing pathogens based on their ability to adhere to intestinal epithelium via actin-filled membranous protrusions (pedestals). Infection of mice with C. rodentium causes breach of the colonic epithelial barrier, a vigorous Th1 inflammatory response, and colitis. Ultimately, an adaptive immune response leads to clearance of the bacteria. Whereas much is known about the adaptive response to C. rodentium, the role of the innate immune response remains unclear. In this study, we demonstrate for the first time that the TLR adaptor MyD88 is essential for survival and optimal immunity following infection. MyD88(-/-) mice suffer from bacteremia, gangrenous mucosal necrosis, severe colitis, and death following infection. Although an adaptive response occurs, MyD88-dependent signaling is necessary for efficient clearance of the pathogen. Based on reciprocal bone marrow transplants in conjunction with assessment of intestinal mucosal pathology, repair, and cytokine production, our findings suggest a model in which TLR signaling in hemopoietic and nonhemopoietic cells mediate three distinct processes: 1) induction of an epithelial repair response that maintains the protective barrier and limits access of bacteria to the lamina propria; 2) production of KC or other chemokines that attract neutrophils and thus facilitate killing of bacteria; and 3) efficient activation of an adaptive response that facilitates Ab-mediated clearance of the infection. Taken together, these experiments provide evidence for a protective role of innate immune signaling in infections caused by attaching and effacing pathogens.     

Changes in brain levels of N-acylethanolamines and 2-arachidonoylglycerol in focal cerebral ischemia in mice

The N -acylethanolamines (NAEs) and 2-arachidonoylglycerol (2-AG) are bioactive lipids that can modulate inflammatory responses and protect neurons against glutamatergic excitotoxicity. We have used a model of focal cerebral ischemia in young adult mice to investigate the relationship between focal cerebral ischemia and endogenous NAEs. Over the first 24 h after induction of permanent middle cerebral artery occlusion, we observed a time-dependent increase in all the investigated NAEs, except for anandamide. Moreover, we found an accumulation of 2-AG at 4 h that returned to basal level 12 h after induction of ischemia. Accumulation of NAEs did not depend on regulation of N -acylphosphatidylethanolamine-hydrolyzing phospholipase D or fatty acid amide hydrolase. Treatment with the fatty acid amide hydrolase inhibitor URB597 (cyclohexyl carbamic acid 3'-carbamoyl-biphenyl-3-yl ester; 1 mg/kg; i.p.) 1.5 h before arterial occlusion decreased the infarct volume in our model system. Our results suggest that NAEs and 2-AG may be involved in regulation of neuroprotection during focal cerebral ischemia in mice.