Fine scale interspersion of conserved sequences in the pea and corn chloroplast genomes

Summary A comparison is made of the chloroplast genomes of two divergent higher plants, pea and corn. Reassociation kinetics analysis shows that only 33–34% of the chloroplast DNA (ct DNA) sequences are conserved in these two plants, which is equal to about 43 kilobases (kb). The restriction enzyme patterns produced by Eco RI, Bam HI, and Sal I are different for each ct DNA, as expected from the low level of homology. The total length of cross-reacting Eco RI fragments, assessed by blot hybridization methods, exceeds the reassociation kinetics estimate by at least 20 kb. An electron microscopic analysis of ct DNA heteroduplexes shows that the conserved regions are surprisingly short, and consequently, they are interspersed with divergent DNA. Fifty percent of the conserved regions are less than 550 bases; 10 sites are less than 150 bases. The median length of a heterologous region is 250 bases. The heteroduplexes fall into 4 classes, established by the position and size of the conserved and divergent regions, totaling 61 kb. One class has been identified as the ribosomal gene region: the corn Eco RI fragment, Eco RI A, which codes for the 16S and 23S cistrons (Bedbrook and Bogorad 1976), was reassociated with total pea ct DNA, and the products analyzed by electron microscopy. Only one pattern of heteroduplexes was observed. A stretch of almost completely conserved DNA, equivalent to 6.9 kb, extends from the 16S gene through the 23S gene, and therefore, includes the transcribed spacer separating these two cistrons. Heterologous regions occur immediately outside of the 5 and 3 ends of the 16S and 23S genes, respectively. A set of the 16S and 23S genes contribute about 4%, and the spacer 1.6%, to the level of sequence homology in each genome.G.K.L. was supported by a National Research Service Award from the National Institutes of Health (GM 07270). This work was also supported by National Institutes of Health Grant GM 22870, and a Grant-in-Aid of Research to G.K.L. from Sigma Xi, The Scientific Research Society     

Allelopathic compounds,thelypterin A and B in the fern Thelypteris normalis

Summary The growth of Thelypteris normalis (C. Chr.) Moxley gametophytes is inhibited under T. normalis sporophytes. Competition for minerals, light, change in pH, or microbial inhibitors were experimentally eliminated as causes of the inhibition. This is the first demonstration of allelopathy between a sporophyte and gametophyte in a fern. Two inhibitors, thelypterin A and B, which were released from the roots of the Thelypteris sporophyte, were isolated and a bioassay for the inhibitors was devised. Thelypterin A gave an Ehrlich-positive reaction indicative of secondary aromatic amines and an ultraviolet absorption spectrum indicative of a heterocyclic ring. The inhibitors affected the growth of Thelypteris, Pteris and Phlebodium gametophytes.     

In situ hatching of Artemia monica cysts in hypersaline Mono Lake,California

Two emergence trap designs were tested in Mono Lake, California, to measure in situ hatching of Artemia monica cysts on the lake bottom. One design incorporated a removable sample bottle; the other had a catch tube which was pumped from the surface. Both traps rested on the bottom and had a narrow gap between the collecting funnel and bottom flange to allow the chemical conditions within the trap to be similar to those outside. This gap was open during April and May but, because some animals entered from outside the area enclosed by the trap, the gap was covered with 400 µm or 800 µm screen during June and July. The two trap types without screens sampled a station in oxic water 7 m deep similarly in April and May 1985. Mean daily hatching rates from April to May 1985 ranged from 720 to 25 340 shrimp m^-2 day^-1. In contrast, mean daily hatching rates during the same period at a station in anoxic water 21 m deep were from 3 to 138 shrimp m^-2 day^-1. June and July hatching rates in the shallow station were lower than in the spring, usually less than 1000 shrimp m^-2 day^-1.     

MIF-I suppresses deprivation-induced fluid consumption in rats

Rats were injected IP with a 0.1 mg/kg dose of MIF-I, naloxone, dynorphin, [D-Phe⁴]-Met-enkephalin, [D-Ala², F₅Phe⁴]-Met-enkephalin-NH₂, or the diluent vehicle, placed in their home cages for ten minutes, and then given ad lib access to either 20% sucrose, 10% sucrose, water, 0.01% quinine, or 0.02% quinine in a repeated measures design with solutions counter-balanced over five days. Fluid consumption was measured every hour for 4 hours. A mixed analysis of variance yielded significant results for all main effects and the peptides by fluid and hours by fluid interactions. For the 4-hr test period, naloxone and [D-Phe⁴]-Met-enkephalin produced reliable increases in consumption while MIF-I produced a reliable decrease. Differences were obtained only with sucrose solutions, and the results clearly suggest that peptides modulate fluid consumption at positive levels of incentive motivation. To reconcile the findings of increased consumption after naloxone with the many studies suggesting a decrease in such paradigms, 0.1, 1.0, and 10.0 mg/kg of naloxone and MIF-I were administered as before but to independent groups of rats and intake was measured every 30 min. These results replicate and extend the above findings by showing that during the first 30-min period, both naloxone and MIF-I suppressed intake in a dose-dependent fashion, with MIF-I being more effective at each dose. The 0.1 mg/kg naloxone group, however, increased consumption over time and achieved a total consumption greater than control animals but comparable to that observed in the first study. It appears that at very low doses naloxone increases consumption over time, but at more commonly tested higher doses it has a suppressant effect. The results support the concept that in many situations MIF-I can produce the same effects as naloxone.     

Development of activity patterns,social interactions,and exploratory behavior in infant tufted capuchins (Cebus apella)

Early organization of activity states was studied in 17 tufted capuchin (Cebus apella) infants from birth to 11 weeks of age. Development of exploration and interactions with mothers and other group members were studied in 14 of these infants up to the age of 1 year. Activity profiles changed from 3 to 8 weeks as infants began to move off mothers and explore their environments. From 2 to 6 months time with mothers decreased; time alone increased correspondingly. Time spent with other group members did not vary significantly over the first year. By 7–9 months capuchin infants spent more time alone or with other group members than with mothers, although weaning was still not completed by the end of the first year. Simple environmental exploration began in the 2nd month and reached stable levels by 4 months. Complex manipulation of food and objects first began at 3–4 months and increased to stable levels in the second half of the first year. Some preliminary differences were evident between infants living in indoor cages and those living in indoor/ outdoor runs. Infants in cages spent less time in dorsal contact with mothers, and less time in social play and proximity to other animals than those in runs. Instead, infants in cages spent more time alone and engaged in more manipulation of food. Some measures of social and exploratory behavior showed a high degree of variability which may be useful in exploring individual differences in infant temperament or reactivity. © 1995 Wiley-Liss, Inc.     

Ubiquitin trafficking to the lysosome: keeping the house tidy and getting rid of unwanted guests

Bacterial killing by autophagic delivery to the lysosomal compartment has been shown for Mycobacteria, Streptococcus, Shigella, Legionella and Salmonella, indicating an important role for this conserved trafficking pathway for the control of intracellular bacterial pathogens.(1-5) In a recent study we found that solubilized lysosomes isolated from bone marrow-derived macrophages had potent antibacterial properties against M. tuberculosis and M. smegmatis that were associated with ubiquitin and ubiquitin-derived peptides. We propose that ubiquitinated proteins are delivered to the lysosomal compartment, where degradation by lysosomal proteinases generates ubiquitin-derived peptides with antimycobacterial properties. This surprising finding provokes a number of questions regarding the nature and trafficking of ubiquitin and ubiquitin-modified proteins in mammalian cells. We discuss the possible role(s) that the multivesicular body (MVB), the late endosome and the autophagosome may play in trafficking of ubiquitinated proteins to the lysosome.     

Riverbed methanotrophy sustained by high carbon conversion efficiency

Our understanding of the role of freshwaters in the global carbon cycle is being revised, but there is still a lack of data, especially for the cycling of methane, in rivers and streams. Unravelling the role of methanotrophy is key to determining the fate of methane in rivers. Here we focus on the carbon conversion efficiency (CCE) of methanotrophy, that is, how much organic carbon is produced per mole of CH₄ oxidised, and how this is influenced by variation in methanotroph communities. First, we show that the CCE of riverbed methanotrophs is consistently high (~50%) across a wide range of methane concentrations (~10–7000 nM) and despite a 10-fold span in the rate of methane oxidation. Then, we show that this high conversion efficiency is largely conserved (50%± confidence interval 44–56%) across pronounced variation in the key functional gene (70 operational taxonomic units (OTUs)), particulate methane monooxygenase (pmoA), and marked shifts in the abundance of Type I and Type II methanotrophs in eight replicate chalk streams. These data may suggest a degree of functional redundancy within the variable methanotroph community inhabiting these streams and that some of the variation in pmoA may reflect a suite of enzymes of different methane affinities which enables such a large range of methane concentrations to be oxidised. The latter, coupled to their high CCE, enables the methanotrophs to sustain net production throughout the year, regardless of the marked temporal and spatial changes that occur in methane.     

Human apolipoprotein E genotypes differentially modify house dust mite-induced airway disease in mice

Apolipoprotein E (apoE) is an endogenous negative regulator of airway hyperreactivity (AHR) and mucous cell metaplasia in experimental models of house dust mite (HDM)-induced airway disease. The gene encoding human apoE is polymorphic, with three common alleles (ε2, ε3, and ε4) reflecting single amino acid substitutions at amino acids 112 and 158. The objective of this study was to assess whether the human apoE alleles modify airway responses to repeated nasal HDM challenges. Mice expressing the human apoE ε2 (huApoE2), ε3 (huApoE3), or ε4 (huApoE4) alleles received nasal HDM challenges, and airway responses were compared with mice expressing the endogenous murine apoE gene (muApoE). huApoE3 mice displayed significant reductions in AHR, mucous cell metaplasia, and airway inflammation compared with muApoE mice. The attenuated severity of airway inflammation in huApoE3 mice was associated with reductions in lung mRNA levels of Th2 and Th17 cytokines, as well as chemokines (CCL7, CCL11, CCL24). huApoE4 mice had an intermediate phenotype, with attenuated AHR and IgE production, compared with muApoE mice, whereas airway inflammation and mucous cell metaplasia were not reduced. In contrast, HDM-induced airway responses were not modified in mice expressing the huApoE2 allele. We conclude that the polymorphic huApoE alleles differentially modulate HDM-induced airway disease, which can be stratified, in rank order of increasing disease severity, ε3 < ε4 < ε2. These results raise the possibility that the polymorphic apoE alleles may modify disease severity in human asthma.     

Altered caveolin-3 expression disrupts PI(3) kinase signaling leading to death of cultured muscle cells

Caveolae and their coat proteins, caveolins, co-ordinate multiple signaling pathways. Caveolin-3 is a muscle-specific caveolin isoform that is deficient in limb girdle muscular dystrophy type 1 C (LGMD1C). Paradoxically, overexpression of this protein also causes muscle degeneration in vivo. We hypothesize that altered membrane expression of caveolin-3 in muscle cells causes a degenerative phenotype by disrupting the co-ordination of signaling pathways that are critical to the maintenance of cell survival. Here, we show for the first time that, in normal muscle cells subjected to oxidative stress, the phosphatidylinositol (3) kinase (PI(3) kinase)-associated proteins PDK1 and Akt associate with caveolae where they bind to caveolin-3, and that normal activation of this pathway promotes cell survival. Either increased or decreased expression of caveolin-3 at the membrane caused an increased susceptibility to oxidative stress, and myotube survival was markedly improved by PI(3) kinase inhibition. This occurred concomitantly with altered phosphorylation of the pro-apoptotic proteins GSK3beta and Bad, despite normal levels of Akt activation. Taken together, our results demonstrate that altered caveolin-3 expression can change the outcome of PI(3) kinase activation from cell survival to cell death. These findings indicate that normal expression and localization of caveolin-3 are required to appropriately co-ordinate PI(3) kinase/Akt-mediated cell survival signaling, and suggest that this pathway may be an effective therapeutic target for the treatment of muscular dystrophies associated with caveolin-3 mutations.     

IL-15-dependent induction of 4-1BB promotes antigen-independent CD8 memory T cell survival

Mice lacking CD137L (4-1BBL) show normal primary expansion and contraction of the CD8+ T cell response to influenza virus, but exhibit a defect in Ag-specific CD8+ T cell numbers at 3-6 wk postinfection. Previous results showed that the decrease in CD8+ T cell numbers in this model is not due to a programming defect during primary expansion. Thus, it appears that 4-1BB/4-1BBL interactions control the number of surviving CD8+ effector memory cells, late in the primary response. In this report, we asked how 4-1BB on T cells could play a role after Ag has apparently been cleared from the host. We show that IL-15, a cytokine involved in regulation of CD8+ memory T cell survival, induces the expression of 4-1BB on CD8+CD44(high) memory phenotype T cells, but not on CD4+ T cells. The Ag-independent induction of 4-1BB by IL-15 was dependent on MAPK p38 and ERK activation. Transfer of in vitro-generated OT-I CD8+ memory T cells into unimmunized wild-type or 4-1BBL-deficient hosts revealed a 2- to 3-fold survival advantage when 4-1BBL was present, recapitulating the effect seen in the endogenous response to influenza in mice. Decreases in the overall number of memory CD8+ T cells were also observed in the bone marrow of unmanipulated 4-1BBL-deficient mice. These data suggest a model whereby 4-1BB expression on memory CD8+ T cells, perhaps due to encounter with IL-15 in the bone marrow, allows 4-1BB/4-1BBL interactions to maintain memory CD8 T cell survival in the absence of Ag.