Molecular cloning of the Haemophilus influenzae gmhA (lpcA) gene encoding a phosphoheptose isomerase required for lipooligosaccharide biosynthesis

We have determined that gene HI#1181 of Haemophilus influenzae is a homolog of Escherichia coli gmhA (previously designated lpcA) (J. S. Brooke and M. A. Valvano, J. Biol. Chem. 271:3608-3614, 1996), which encodes a phosphoheptose isomerase catalyzing the first step of the biosynthesis of ADP-L-glycero-D-manno heptose. Mutations in this gene are associated with a heptoseless core lipopolysaccharide which determines an increased outer membrane permeability to hydrophobic compounds. The cloned H. influenzae gmhA restored the synthesis of a complete core in the gmhA-deleted E. coli strain chi711. Amino acid sequence comparisons of the GmhA proteins of E. coli and H. influenzae with other proteins in the databases revealed the existence of a novel family of phosphosugar a1do-keto isomerases.

Two-dimensional Monte Carlo simulations of H i line formation in massive young stellar object disc winds

Massive young stellar objects (YSOs) are powerful infrared Hi line emitters. It has been suggested that these lines form in an outflow from a disc surrounding the YSO. Here, new two-dimensional Monte Carlo radiative transfer calculations are described which test this hypothesis. Infrared spectra are synthesized for a YSO disc wind model based on earlier hydrodynamical calculations. The model spectra are in qualitative agreement with the observed spectra from massive YSOs, and therefore provide support for a disc wind explanation for the Hi lines. However, there are some significant differences: the models tend to overpredict the Bra/Br? ratio of equivalent widths and produce line profiles which are slightly too broad and, in contrast to typical observations, are double-peaked. The interpretation of these differences within the context of the disc wind picture and suggestions for their resolution via modifications to the assumed disc and outflow structure are discussed. © 2005 RAS.

Quantification of malondialdehyde and 4-hydroxynonenal adducts to lysine residues in native and oxidized human low-density lipoprotein

Malondialdehyde (MDA) and 4-hydroxynonenal (HNE) are major end-products of oxidation of polyunsaturated fatty acids, and are frequently measured as indicators of lipid peroxidation and oxidative stress in vivo. MDA forms Schiff-base adducts with lysine residues and cross-links proteins in vitro; HNE also reacts with lysines, primarily via a Michael addition reaction. We have developed methods using NaBH4 reduction to stabilize these adducts to conditions used for acid hydrolysis of protein, and have prepared reduced forms of lysine-MDA [3-(N epsilon-lysino)propan-1-ol (LM)], the lysine-MDA-lysine iminopropene cross-link [1,3-di(N epsilon-lysino)propane (LML)] and lysine-HNE [3-(N epsilon-lysino)-4-hydroxynonan-l-ol (LHNE)]. Gas chromatography/MS assays have been developed for quantification of the reduced compounds in protein. RNase incubated with MDA or HNE was used as a model for quantification of the adducts by gas chromatography/MS. There was excellent agreement between measurement of MDA bound to RNase as LM and LML, and as thiobarbituric acid-MDA adducts measured by HPLC; these adducts accounted for 70-80% of total lysine loss during the reaction with MDA. LM and LML (0.002-0.12 mmol/ mol of lysine) were also found in freshly isolated low-density lipoprotein (LDL) from healthy subjects. LHNE was measured in RNase treated with HNE, but was not detectable in native LDL. LM, LML and LHNE increased in concert with the formation of conjugated dienes during the copper-catalysed oxidation of LDL, but accounted for modification of <1% of lysine residues in oxidized LDL. These results are the first report of direct chemical measurement of MDA and HNE adducts to lysine residues in LDL. LM, LML and LHNE should be useful as biomarkers of lipid peroxidative modification of protein and of oxidative stress in vitro and in vivo.

The advanced glycation end product, Nepsilon-(carboxymethyl)lysine, is a product of both lipid peroxidation and glycoxidation reactions

Nepsilon-(Carboxymethyl)lysine (CML) is an advanced glycation end product formed on protein by combined nonenzymatic glycation and oxidation (glycoxidation) reactions. We now report that CML is also formed during metal-catalyzed oxidation of polyunsaturated fatty acids in the presence of protein. During copper-catalyzed oxidation in vitro, the CML content of low density lipoprotein increased in concert with conjugated dienes but was independent of the presence of the Amadori compound, fructoselysine, on the protein. CML was also formed in a time-dependent manner in RNase incubated under aerobic conditions in phosphate buffer containing arachidonate or linoleate; only trace amounts of CML were formed from oleate. After 6 days of incubation the yield of CML in RNase from arachidonate was approximately 0.7 mmol/mol lysine compared with only 0.03 mmol/mol lysine for protein incubated under the same conditions with glucose. Glyoxal, a known precursor of CML, was also formed during incubation of RNase with arachidonate. These results suggest that lipid peroxidation, as well as glycoxidation, may be an important source of CML in tissue proteins in vivo and that CML may be a general marker of oxidative stress and long term damage to protein in aging, atherosclerosis, and diabetes.

Multi-epoch high-resolution spectroscopy of SN2011fe - Linking the progenitor to its environment

This study attempts to establish a link between the reasonably well known nature of the progenitor of SN2011fe and its surrounding environment. This is done with the aim of enabling the identification of similar systems in the vast majority of the cases, when distance and epoch of discovery do not allow a direct approach. To study the circumstellar environment of SN2011fe we have obtained high-resolution spectroscopy of SN2011fe on 12 epochs, from 8 to 86 days after the estimated date of explosion, targeting in particular at the time evolution of CaII and NaI. Three main absorption systems are identified from CaII and NaI, one associated to the Milky Way, one probably arising within a high-velocity cloud, and one most likely associated to the halo of M101. The Galactic and host galaxy reddening, deduced from the integrated equivalent widths (EW) of the NaI lines are E(B-V)=0.011+/-0.002 and E(B-V)=0.014+/-0.002 mag, respectively. The host galaxy absorption is dominated by a component detected at the same velocity measured from the 21-cm HI line at the projected SN position (~180 km/s). During the ~3 months covered by our observations, its EW changed by 15.6+/-6.5 mA. This small variation is shown to be compatible with the geometric effects produced by therapid SN photosphere expansion coupled to the patchy fractal structure of the ISM. The observed behavior is fully consistent with ISM properties similar to those derived for our own Galaxy, with evidences for structures on scales

Isotretinoin therapy changes the expression of antimicrobial peptides in acne vulgaris

In acne vulgaris, antimicrobial peptides (AMPs) could play a dual role; i.e., protective by acting against Propionibacterium acnes, pro-inflammatory by acting as signalling molecules. The cutaneous expression of 15 different AMPs was investigated in acne patients; furthermore, the impact of isotretinoin therapy on AMP expression was analysed in skin biopsies from 13 patients with acne vulgaris taken before, during and after a 6-month treatment cycle with isotretinoin using quantitative real-time polymerase chain reaction. Cutaneous expression of the AMPs cathelicidin, human β-defensin-2 (HBD-2), lactoferrin, lysozyme, psoriasin (S100A7), koebnerisin (S100A15), and RNase 7 was upregulated in untreated acne vulgaris, whereas α-defensin-1 (HNP-1) was downregulated compared to controls. While relative expression levels of cathelicidin, HBD-2, lactoferrin, psoriasin (S100A7), and koebnerisin (S100A15) decreased during isotretinoin treatment, only those of cathelicidin and koebnerisin returned to normal after 6 months of isotretinoin therapy. The increased expression of lysozyme and RNase 7 remained unaffected by isotretinoin treatment. The levels of granulysin, RANTES (CCL5), perforin, CXCL9, substance P, chromogranin B, and dermcidin were not regulated in untreated acne patients and isotretinoin had no effect on these AMPs. In conclusion, the expression of various AMPs is altered in acne vulgaris. Isotretinoin therapy normalizes the cutaneous production of distinct AMPs while the expression of others is still increased in healing acne. Considering the antimicrobial and pro-inflammatory role of AMPs, these molecules could serve as specific targets for acne therapy and maintenance of clinical remission.

Line-driven radiative outflows in luminous quasars

An analysis of ≃19 500 narrow (≲200 km s^-1) CIV λλ1548.2,1550.8 absorbers in ≃34 000 Sloan Digital Sky Survey quasar spectra is presented. The statistics of the number of absorbers as a function of outflow velocity shows that in approximately two-thirds of outflows, with multiple C IV absorbers present, absorbers are line-locked at the 500 km s^-1 velocity separation of the C IV absorber doublet; appearing as 'triplets' in the quasar spectra. Line-locking is an observational signature of radiative line-driving in outflowing material, where the successive shielding of 'clouds' of material in the outflow locks the clouds together in outflow velocity. Line-locked absorbers are seen in both broad absorption line (BAL) quasars and non-BAL quasars with comparable frequencies and with velocities out to at least 20 000 km s^-1. There are no detectable differences in the absorber properties and the dust content of single C IV doublets and line-locked C IV doublets. The gas associated with both single and line-locked CIV absorption systems includes material with a wide range of ionization potential (14-138 eV). Both single and line-locked CIV absorber systems show strong systematic trends in their ionization as a function of outflow velocity, with ionization decreasing rapidly with increasing outflow velocity. Initial simulations, employing CLOUDY, demonstrate that a rich spectrum of line-locked signals at various velocities may be expected due to significant opacities from resonance lines of Li-, He- and H-like ions of O, C and N, along with contributions from He II and HI resonance lines. The simulations confirm that line-driving can be the dominant acceleration mechanism for clouds with N(H I) ≃ 10¹⁹ cm^-2.

Tonnacypris glacialis (Ostracoda, Cyprididae):taxonomic position, (palaeo)ecology, and zoogeography

The freshwater ostracod Tonnacypris glaciallis (Sars, 1890) is reported from the European Pleistocene for the first time. The historical allocation of the species is discussed, and the species composition and characteristics of Tonnacypris is Diebel & Pietrzeniuk (1975) and its phylozoogeography are considered. The significance of T. glacialis is reviewed, particularly from the viewpoint of the possible implications of parthenogenesis (and occasional-male production) for the Quaternary history of the genus, and for the use of the species in palaeoenvironmental reconstruction. It is suggested that the Pleistocene fossil occurrence of I: glacialis in modern temperate latitudes is a robust indicator of mean summer temperatures of 6 degrees C.

The Bdellovibrio bacteriovorus twin-arginine transport system has roles in predatory and prey-independent growth

Bdellovibrio bacteriovorus grows in one of two ways: either (i) predatorily [in a host-dependent (HD) manner], when it invades the periplasm of another Gram-negative bacterium, exporting into the prey co-ordinated waves of soluble enzymes using the prey cell contents for growth; or (ii) in a host-independent (HI) manner, when it grows (slowly) axenically in rich media. Periplasmic invasion potentially exposes B. bacteriovorus to extremes of pH and exposes the need to scavenge electron donors from prey electron transport components by synthesis of metalloenzymes. The twin-arginine transport system (Tat) in other bacteria transports folded metalloenzymes and the B. bacteriovorus genome encodes 21 potential Tat-transported substrates and Tat transporter proteins TatA1, TatA2 and TatBC. GFP tagging of the Tat signal peptide from Bd1802, a high-potential iron-sulfur protein (HiPIP), revealed it to be exported into the prey bacterium during predatory growth. Mutagenesis showed that the B. bacteriovorus tatA2 and tatC gene products are essential for both HI and HD growth, despite the fact that they partially complement (in SDS resistance assays) the corresponding mutations in Escherichia coli where neither TatA nor TatC are essential for life. The essentiality of B. bacteriovorus TatA2 was surprising given that the B. bacteriovorus genome encodes a second tatA homologue, tatA1. Transcription of tatA1 was found to be induced upon entry to the bdelloplast, and insertional inactivation of tatA1 showed that it significantly slowed the rates of both HI and HD growth. B. bacteriovorus is one of a few bacterial species that are reliant on a functional Tat system and where deletion of a single tatA1 gene causes a significant growth defect(s), despite the presence of its tatA2 homologue.

Three motAB stator gene products in Bdellovibrio bacteriovorus contribute to motility of a single flagellum during predatory and prey-independent growth

The predatory bacterium Bdellovibrio bacteriovorus uses flagellar motility to locate regions rich in Gram-negative prey bacteria, colliding and attaching to prey and then ceasing flagellar motility. Prey are then invaded to form a "bdelloplast" in a type IV pilus-dependent process, and prey contents are digested, allowing Bdellovibrio growth and septation. After septation, Bdellovibrio flagellar motility resumes inside the prey bdelloplast prior to its lysis and escape of Bdellovibrio progeny. Bdellovibrio can also grow slowly outside prey as long flagellate host-independent (HI) cells, cultured on peptone-rich media. The B. bacteriovorus HD100 genome encodes three pairs of MotAB flagellar motor proteins, each of which could potentially form an inner membrane ion channel, interact with the FliG flagellar rotor ring, and produce flagellar rotation. In 2004, Flannagan and coworkers (R. S. Flannagan, M. A. Valvano, and S. F. Koval, Microbiology 150:649-656, 2004) used antisense RNA and green fluorescent protein (GFP) expression to downregulate a single Bdellovibrio motA gene and reported slowed release from the bdelloplast and altered motility of the progeny. Here we inactivated each pair of motAB genes and found that each pair contributes to motility, both predatorily, inside the bdelloplast and during HI growth; however, each pair was dispensable, and deletion of no pair abolished motility totally. Driving-ion studies with phenamil, carbonyl cyanide m-chlorophenylhydrazone (CCCP), and different pH and sodium conditions indicated that all Mot pairs are proton driven, although the sequence similarities of each Mot pair suggests that some may originate from halophilic species. Thus, Bdellovibrio is a "dedicated motorist," retaining and expressing three pairs of mot genes.

Alterations in Circulating Immune Cells in Neovascular Age-Related Macular Degeneration

Neovascular age-related macular degeneration (nAMD) is the leading cause of irreversible blindness in developed countries. Recent advances have highlighted the essential role of inflammation in the development of the disease. In addition to local retinal chronic inflammatory response, systemic immune alterations have also been observed in AMD patients. In this study we investigated the association between the frequency of circulating leukocyte populations and the prevalence as well as clinical presentations of nAMD. Leukocyte subsets of 103 nAMD patients (most of them were receiving anti-VEGF therapy prior to enrolment) and 26 controls were analysed by flow cytometry by relative cell size, granularity and surface markers. Circulating CD11b(+) cells and CD16(hi)HLA-DR(-) neutrophils were significantly increased (P = 0.015 and 0.009 respectively) in nAMD when compared to controls. The percentage of circulating CD4(+) T-cells was reduced in nAMD patients without subretinal fibrosis (P = 0.026) compared to patients with subretinal fibrosis. There was no correlation between the percentage of circulating leukocytes and the responsiveness to anti-VEGF therapy in nAMD patients. Our results suggest that higher levels of circulating CD11b(+) cells and neutrophils are associated with nAMD and that reduced levels of CD4(+) T-cells are associated with the absence of subretinal fibrosis in nAMD.

Two-part Segmentation of Text Documents

We consider the problem of segmenting text documents that have a
two-part structure such as a problem part and a solution part. Documents
of this genre include incident reports that typically involve
description of events relating to a problem followed by those pertaining
to the solution that was tried. Segmenting such documents
into the component two parts would render them usable in knowledge
reuse frameworks such as Case-Based Reasoning. This segmentation
problem presents a hard case for traditional text segmentation
due to the lexical inter-relatedness of the segments. We develop
a two-part segmentation technique that can harness a corpus
of similar documents to model the behavior of the two segments
and their inter-relatedness using language models and translation
models respectively. In particular, we use separate language models
for the problem and solution segment types, whereas the interrelatedness
between segment types is modeled using an IBM Model
1 translation model. We model documents as being generated starting
from the problem part that comprises of words sampled from
the problem language model, followed by the solution part whose
words are sampled either from the solution language model or from
a translation model conditioned on the words already chosen in the
problem part. We show, through an extensive set of experiments on
real-world data, that our approach outperforms the state-of-the-art
text segmentation algorithms in the accuracy of segmentation, and
that such improved accuracy translates well to improved usability
in Case-based Reasoning systems. We also analyze the robustness
of our technique to varying amounts and types of noise and empirically
illustrate that our technique is quite noise tolerant, and
degrades gracefully with increasing amounts of noise