Effect of tagetitoxin on the levels of ribulose 1,5-bisphosphate carboxylase, ribosomes, and RNA in plastids of wheat leaves

Growth of wheat seedlings in the presence of the phytotoxin tagetitoxin produces pigment-deficient leaves of normal size and morphology whose cells contain only rudimentary plastids. We could not detect the accumulation of either the plastid-encoded large subunit or the nuclear-encoded small subunit of the chloroplast stromal enzyme ribulose 1,5-bisphosphate carboxylase (RuBPCase) in western blots of protein extracted from leaves of such seedlings. Sucrose gradient centrifugation profiles showed that plastid ribosomes were essentially absent in toxin-treated leaf tissue while cytoplasmic ribosomes were relatively unaffected. Northern blot analysis of RNA in toxin-treated leaves showed a deficiency of plastid ribosomal RNA (16S and 23S) as well as reduced levels of plastid mRNAs for the large subunit of RuBPCase and for the 32 kilodalton thylakoid Q_B polypeptide. Northern analysis also showed that the nuclear-encoded rbcS mRNA for the small subunit of RuBPCase is present in only trace amounts in toxin-treated leaves.     

Collagen polysomes: Site of hydroxylation of proline residues

The biosynthesis of collagen on polysomes has been studied by using a newly devised method for obtaining polysomes in high yield from stationary-phase mouse fibroblast (line 3T6; Goldberg &, Green, 1967). These polysomes were completely disaggregated to monosomes by brief exposure to ribonuclease and they lost most of their radioactivity to the top of the sucrose gradients as a result of a 30-minute chase with unlabeled proline. After a ten-minute pulse with [³H]proline, nascent collagen peptides could be identified in these polysomes on sucrose gradients. Most of the proline residues susceptible to hydroxylation by collagen proline hydroxylase were found, in most cases, to be already hydroxylated in these nascent peptides. The nascent nature of these peptides was confirmed by the observation that treatment of the polysomes with RNase transferred the radioactive collagen peptides to the monosome area and these peptides could subsequently be removed to the soluble material at the top of the gradient upon treatment with puromycin. These findings therefore, show clearly that the hydroxylation of proline residues is occurring, in vivo under normal conditions, on nascent collagen chains. In no case was the degree of hydroxylation of the released collagen chains higher than that on the nascent collagen peptides. It seems likely, therefore, that the major site of proline hydroxylation is the nascent collagen peptide.     

SAA-only HDL formed during the acute phase response in apoA-I+/+ and apoA-I-/- mice.

Serum amyloid A (SAA) is an acute phase protein of unknown function that is involved in systemic amyloidosis and may also be involved in atherogenesis. The precise role of SAA in these processes has not been established. SAA circulates in plasma bound to high density lipoprotein-3 (HDL3). The pathway for the production of SAA-containing HDL is not known. To test whether apolipoprotein (apo)A-I-HDL is required in the production of SAA-HDL, we analyzed the lipopolysaccharide (LPS)-induced changes in apoA-I+/+ and apoA-I-/- mice. In apoA-I+/+ mice, after injection of LPS, remodeling of HDL occurred: total cholesterol increased and apoA-I decreased slightly and shifted to lighter density. Dense (density of HDL3) but large (size of HDL2 ) SAA-containing particles were formed. Upon fast phase liquid chromatography fractionation of plasma, >90% of SAA eluted with HDL that was enriched in cholesterol and phospholipid and shifted "leftward" to larger particles. Non-denaturing immunoprecipitation with anti-mouse apoA-I precipitated all of the apoA-I but not all of the SAA, confirming the presence of SAA-HDL devoid of apoA-I. In the apoA-I-/- mice, which normally have very low plasma lipid levels, LPS injection resulted in significantly increased total and HDL cholesterol. Greater than 90% of the SAA was lipid associated and was found on dense but large, spherical HDL particles essentially devoid of other apolipoproteins.We conclude that serum amyloid A (SAA) is able to sequester lipid, forming dense but large HDL particles with or without apoA-I or other apolipoproteins. The capacity to isolate lipoprotein particles containing SAA as the predominant or only apolipoprotein provides an important system to further explore the biological function of SAA.     

Segmental structure of the Brassica napus genome based on comparative analysis with Arabidopsis thaliana

Over 1000 genetically linked RFLP loci in Brassica napus were mapped to homologous positions in the Arabidopsis genome on the basis of sequence similarity. Blocks of genetically linked loci in B. napus frequently corresponded to physically linked markers in Arabidopsis. This comparative analysis allowed the identification of a minimum of 21 conserved genomic units within the Arabidopsis genome, which can be duplicated and rearranged to generate the present-day B. napus genome. The conserved regions extended over lengths as great as 50 cM in the B. napus genetic map, equivalent to approximately 9 Mb of contiguous sequence in the Arabidopsis genome. There was also evidence for conservation of chromosome landmarks, particularly centromeric regions, between the two species. The observed segmental structure of the Brassica genome strongly suggests that the extant Brassica diploid species evolved from a hexaploid ancestor. The comparative map assists in exploiting the Arabidopsis genomic sequence for marker and candidate gene identification within the larger, intractable genomes of the Brassica polyploids.     

Patterns of sequence loss and cytosine methylation within a population of newly resynthesized Brassica napus allopolyploids

Allopolyploid formation requires the adaptation of two nuclear genomes within a single cytoplasm, which may involve programmed genetic and epigenetic changes during the initial generations following genome fusion. To study the dynamics of genome change, we synthesized 49 isogenic Brassica napus allopolyploids and surveyed them with 76 restriction fragment length polymorphism (RFLP) probes and 30 simple sequence repeat (SSR) primer pairs. Here, we report on the types and distribution of genetic and epigenetic changes within the S(1) genotypes. We found that insertion/deletion (indel) events were rare, but not random. Of the 57,710 (54,383 RFLP and 3,327 SSR) parental fragments expected among the amphidiploids, we observed 56,676 or 99.9%. Three loci derived from Brassica rapa had indels, and one indel occurred repeatedly across 29% (14/49) of the lines. Loss of one parental fragment was due to the 400-bp reduction of a guanine-adenine dinucleotide repeat-rich sequence. In contrast to the 4% (3/76) RFLP probes that detected indels, 48% (35/73) detected changes in the CpG methylation status between parental genomes and the S1 lines. Some loci were far more likely than others to undergo epigenetic change, but the number of methylation changes within each synthetic polyploid was remarkably similar to others. Clear de novo methylation occurred at a much higher frequency than de novo demethylation within allopolyploid sequences derived from B. rapa. Our results suggest that there is little genetic change in the S(0) generation of resynthesized B. napus polyploids. In contrast, DNA methylation was altered extensively in a pattern that indicates tight regulation of epigenetic changes.     

Defining winter trophic habitat of juvenile Gulf Sturgeon in the Suwannee and Apalachicola rivermouth estuaries, acoustic telemetry investigations

Three automated listening post‐telemetry studies were undertaken in the Suwannee and Apalachicola estuaries to gain knowledge of habitats use by juvenile Gulf Sturgeons (Acipenser oxyrinchus desotoi) on winter feeding grounds. A simple and reliable method for external attachment of small acoustic tags to the dorsal fin base was developed using shrink‐tubing. Suspending receivers on masts below anchored buoys improved reception and facilitated downloading; a detection range of 500–2500 m was realized. In the Apalachicola estuary, juvenile GS stayed in shallow water (< 2 m) within the estuarine transition zone all winter in the vicinity of the Apalachicola River mouth. Juvenile GS high‐use areas did not coincide with high density benthic macrofauna areas from the most recent (1999) benthos survey. In the Suwannee estuary, juveniles ranged widely and individually throughout oligohaline to mesohaline subareas of the estuary, preferentially using mesohaline subareas seaward of Suwannee Reef (52% of acoustic detections). The river mouth subarea was important only in early and late winter, during the times of adult Gulf Sturgeon migrations (41% of detections). Preferred winter feeding subareas coincided spatially with known areas of dense macrofaunal benthos concentrations. Following a dramatic drop in air and water temperatures, juvenile GS left the river mouth and estuary, subsequently being detected 8 km offshore in polyhaline open Gulf of Mexico waters, before returning to the estuary. Cold‐event offshore excursions demonstrate that they can tolerate full‐salinity polyhaline waters in the open Gulf of Mexico, for at least several days at a time. For juvenile sturgeons, the stress and metabolic cost of enduring high salinity ( Jarvis et al., 2001 ; McKenzie et al., 2001 ; Singer and Ballantyne, 2002 ) for short periods in deep offshore waters seems adaptively advantageous relative to the risk of cold‐event mortality in shallow inshore waters of lower salinity. Thus, while juveniles can tolerate high salinities for days to weeks to escape cold events, they appear to make only infrequent use of open polyhaline waters. Throughout the winter foraging period, juvenile GS stayed primarily within the core area of Suwannee River mouth influence, extending about 12 km north and south of the river mouth, and somewhat seaward of Suwannee Reef (< 5 km offshore). None were detected departing the core area past either of the northern or southern acoustic gates, located 66 and 52 km distant from the river mouth, respectively.     

Liver Is Able to Activate Na?ve CD8+ T Cells with Dysfunctional Anti-Viral Activity in the Murine System

The liver possesses distinct tolerogenic properties because of continuous exposure to bacterial constituents and nonpathogenic food antigen. The central immune mediators required for the generation of effective immune responses in the liver environment have not been fully elucidated. In this report, we demonstrate that the liver can indeed support effector CD8⁺ T cells during adenovirus infection when the T cells are primed in secondary lymphoid tissues. In contrast, when viral antigen is delivered predominantly to the liver via intravenous (IV) adenovirus infection, intrahepatic CD8⁺ T cells are significantly impaired in their ability to produce inflammatory cytokines and lyse target cells. Additionally, intrahepatic CD8⁺ T cells generated during IV adenovirus infection express elevated levels of PD-1. Notably, lower doses of adenovirus infection do not rescue the impaired effector function of intrahepatic CD8⁺ T cell responses. Instead, intrahepatic antigen recognition limits the generation of potent anti-viral responses at both priming and effector stages of the CD8⁺ T cell response and accounts for the dysfunctional CD8⁺ T cell response observed during IV adenovirus infection. These results also implicate that manipulation of antigen delivery will facilitate the design of improved vaccination strategies to persistent viral infection.