Fluorescence quenching of IsiA in early stage of iron deficiency and at cryogenic temperatures

Cyanobacteria respond to iron deficiency during growth by expressing the isiA gene, which produces a chlorophyll-carotenoid protein complex known as IsiA or CP43′. Long-term iron deficiency results in the formation of large IsiA aggregates, some of which associate with photosystem I (PSI) while others are not connected to a photosystem. The fluorescence at room temperature of these unconnected aggregates is strongly quenched, which points to a photoprotective function. In this study, we report time-resolved fluorescence measurements of IsiA aggregates at low temperatures. The average fluorescence lifetimes are estimated to be about 600 ps at 5 K and 150 ps at 80 K. Both lifetimes are much shorter than that of the monomeric complex CP47 at 77 K. We conclude that IsiA aggregates quench fluorescence to a significant extent at cryogenic temperatures. We show by low-temperature fluorescence spectroscopy that unconnected IsiA is present already after two days of growth in an iron-deficient medium, when PSI and PSII are still present in significant amounts and that under these conditions the fluorescence quenching is similar to that after 18 days, when PSI is almost completely absent. We conclude that unconnected IsiA provides photoprotection in all stages of iron deficiency.     

Expression and Purification of Isotopically Enriched MHC Binding Immunogenic Peptides for NMR Studies

Peptides are important naturally occurring ligands of MHC molecules. X-ray crystallographic studies have enabled extensive characterization of such peptide ligands. Yet structural and dynamic changes of these peptides in the MHC bound state are not well understood. These conformational transitions are key to understanding the function of MHC molecules and for the development of peptide-based therapeutics. Employing NMR for such studies can fill this gap but it requires the availability of peptides labeled with NMR-active nuclei. Here we report production of nine-mer MHC-binding peptides for use in high resolution NMR studies. The method utilizes a fusion protein approach of attaching the peptide to an easily expressed bacterial protein. The fusion protein construct design allows for rapid purification of the fusion protein and avoids chemical modification of the peptide as a result of the cleavage reaction. The methods developed here allow for rapid cloning of additional MHC binding peptides without significant molecular biology effort. 8?C10 mg of mature freeze dried peptides can be obtained from 1 liter of minimal media, sufficient for NMR experimentation. Six uniformly ¹⁵N-labeled peptides have been successfully expressed in bacteria and NMR spectra with the expected number of well-resolved signals were recorded. The results obtained here will make peptide-MHC complexes amenable to structural analysis which has not been possible previously.     

STRUCTURALLY PRESERVED FOSSIL PLANTS FROM ANTARCTICA. I. ANTARCTICYCAS,GEN. NOV., A TRIASSIC CYCAD STEM FROM THE BEARDMORE GLACIER AREA

Silicified stems with typical cycadalean anatomy are described from specimens collected from the Fremouw Formation (Triassic) in the Transantarctic Mountains of Antarctica. Axes are slender with a large parenchymatous pith and cortex separated by a narrow ring of vascular tissue. Mucilage canals are present in both pith and cortex. Vascular tissue consists of endarch primary xylem, a narrow band of secondary xylem tracheids, cambial zone, and region of secondary phloem. Vascular bundles contain uni- to triseriate rays with larger rays up to 2 mm wide separating the individual bundles. Pitting on primary xylem elements ranges from helical to scalariform; secondary xylem tracheids exhibit alternate circular bordered pits. Traces, often accompanied by a mucilage canal, extend out through the large rays into the cortex where some assume a girdling configuration. A zone of periderm is present at the periphery of the stem. Large and small roots are attached to the stem and are conspicuous in the surrounding matrix. The anatomy of the Antarctic cycad is compared with that of other fossil and extant cycadalean stems.     

Leukemia inhibitory factor induces apoptosis of the mammary epithelial cells and participates in mouse mammary gland involution

Leukemia inhibitory factor (LIF) is a multifunctional glycoprotein that displays multiple biological activities in different cell types, but to date there has been no report on its expression in the normal mammary gland. In this study we found that LIF is expressed at low but detectable levels in postpubertal, adult virgin, and pregnant mouse mammary glands. However, LIF expression drops after parturition to become almost undetectable in lactating glands. Interestingly, LIF expression shows a steep increase shortly after weaning that is maintained for the following 3 days. During this period, known as the first stage of mammary gland involution, the lack of suckling induces local factors that cause extensive epithelial cell death. It has been shown that Stat3 is the main factor in signaling the initiation of apoptosis, but the mechanism of its activation remains unclear. Herein, we show that LIF expression in the gland is induced by milk stasis and not by the decrease of circulating lactogenic hormones after weaning. Implantation of LIF containing pellets in lactating glands results in a significant increase in epithelium apoptosis. In addition, this treatment also induces Stat3 phosphorylation. We conclude that LIF regulated expression in the mouse mammary gland may play a relevant role during the first stage of mammary gland involution. Our results also show that LIF-induced mammary epithelium apoptosis could be mediated, at least partially, by Stat3 activation.     

Spatial variation of the stable nitrogen isotope ratio of woody plants along a topoedaphic gradient in a subtropical savanna

Variation in the stable N isotope ratio (δ¹⁵N) of plants and soils often reflects the influence of environment on the N cycle. We measured leaf δ¹⁵N and N concentration ([N]) on all individuals of Prosopis glandulosa (deciduous tree legume), Condalia hookeri (evergreen shrub), and Zanthoxylum fagara (evergreen shrub) present within a belt transect 308 m long × 12 m wide in a subtropical savanna ecosystem in southern Texas, USA in April and August 2005. Soil texture, gravimetric water content (GWC), total N and δ¹⁵N were also measured along the transect. At the landscape scale, leaf δ¹⁵N was negatively related to elevation for all the three species along this topoedaphic sequence. Changes in soil δ¹⁵N, total N, and GWC appeared to contribute to this spatial pattern of leaf δ¹⁵N. In lower portions of the landscape, greater soil N availability and GWC are associated with relatively high rates of both N mineralization and nitrification. Both soil δ¹⁵N and leaf [N] were positively correlated with leaf δ¹⁵N of non-N₂ fixing plants. Leaf δ¹⁵N of P. glandulosa, an N₂-fixing legume, did not correlate with leaf [N]; the δ¹⁵N of P. glandulosa’s leaves were closer to atmospheric N₂ and significantly lower than those of C. hookeri and Z. fagara. Additionally, at smaller spatial scales, a proximity index (which reflected the density and distance of surrounding P. glandulosa trees) was negatively correlated with leaf δ¹⁵N of C. hookeri and Z. fagara, indicating the N₂-fixing P. glandulosa may be important to the N nutrition of nearby non-N₂-fixing species. Our results indicate plant ¹⁵N natural abundance can reflect the extent of N retention and help us better understand N dynamics and plant-soil interactions at ecosystem and landscape scales.     

Deletion of a xenobiotic metabolizing gene in mice affects folate metabolism

The mouse arylamine N-acetyltransferase 2 (Nat2) and its homologue (NAT1) in humans are known to detoxify xenobiotic arylamines and are also thought to play a role in endogenous metabolism. Human NAT1 is highly over-expressed in estrogen receptor positive breast tumours and is implicated in susceptibility to neural tube defects. In vitro assays have suggested an endogenous role for human NAT1 in folate metabolism, but in vivo evidence to support this hypothesis has been lacking. Mouse Nat2 provides a good model to study human NAT1 as it shows similar expression profiles and substrate specificities. We have generated transgenic mice lacking a functional Nat2 gene and compared the urinary levels of acetylated folate metabolite para-aminobenzoylglutamate in Nat2 knockout and Nat2 wild-type mice. These results support an in vivo role for mouse Nat2/human NAT1 in folate metabolism. In addition, effects of the Nat2 deletion on sex ratios and neural tube development are described.     

Secondary phloem anatomy of Cycadeoidea (Bennettitales)

Secondary phloem anatomy of several species of Cycadeoidea is described from trunks in the Wieland Collection, Peabody Museum of Natural History. The trunks were collected from the Lakota Formation, Lower Cretaceous, Black Hills of South Dakota. Secondary phloem is extensively developed and consists of alternating, tangential bands of fibers and sieve elements, with rare phloem parenchyma. Uniseriate rays, 2-22 cells high, occur between every one to three files of the axial system. Fibers are long, more than 1200 μm, approximately 26.6-34.2 μm in diameter, and have slit-like apertures on the lateral walls. Sieve elements range from 16-25 μm in diameter and are up to 500 μm long. Elliptical sieve areas appear on both end and radial walls and measure 10 μm across; minute spots, which may represent sieve pores, are present within the sieve areas. Secondary phloem of North American Cycadeoidea is similar in organization (alternating tangential bands) and cell types (sieve cells, fibers, axial parenchyma) to that known in other extant and fossil cycadophytes and some seed ferns. The unusual pattern of cell types and thickness of secondary phloem is discussed in the context of plant habit, phloem efficiency, and potential phylogenetic importance.     

Inhibition of mycobacterial arylamine N-acetyltransferase contributes to anti-mycobacterial activity of Warburgia salutaris

In this study, we show that extracts and a purified compound of Warburgia salutaris exhibit anti-mycobacterial activity against Mycobacterium tuberculosis H37Rv and Mycobacterium bovis BCG Pasteur. The extracts did not inhibit growth of Escherichia coli and were not toxic to cultured mammalian macrophage cells at the concentrations at which anti-mycobacterial activity was observed. The extract and pure compound inhibited pure recombinant arylamine N-acetyltransferase (NAT), an enzyme involved in mycobacterial cell wall lipid synthesis. Moreover, neither extract nor pure compound inhibited growth of a strain of M. bovis BCG in which nat has been deleted suggesting that NAT may indeed be a target within the mycobacterial cell. The purified compound is a novel drimane sesquiterpenoid lactone, 11alpha-hydroxycinnamosmolide. These studies show that W. salutaris is a useful source of anti-tubercular compounds for further analysis and supports the hypothesis of a link between NAT inhibition and anti-mycobacterial activity.