Over-the-barrier transition state analogues and crystal structure with Mycobacterium tuberculosis purine nucleoside phosphorylase

Stable chemical analogues of enzymatic transition states are imperfect mimics since they lack the partial bond character of the transition state. We synthesized structural variants of the Immucillins as transition state analogues for purine nucleoside phosphorylase and characterized them with the enzyme from Mycobacterium tuberculosis (MtPNP). PNPs form transition states with ribooxacarbenium ion character and catalyze nucleophilic displacement reactions by migration of the cationic ribooxacarbenium carbon between the enzymatically immobilized purine and phosphate nucleophiles. As bond-breaking progresses, carbocation character builds on the ribosyl group, the distance between the purine and the carbocation increases, and the distance between carbocation and phosphate anion decreases. Transition state analogues were produced with carbocation character and increased distance between the ribooxacarbenium ion and the purine mimics by incorporating a methylene bridge between these groups. Immucillin-H (ImmH), DADMe-ImmH, and DADMe-ImmG mimic the transition state of MtPNP and are slow-onset, tight-binding inhibitors of MtPNP with equilibrium dissociation constants of 650, 42, and 24 pM. Crystal structures of MtPNP complexes with ImmH and DADMe-ImmH reveal an ion-pair between the inhibitor cation and the nucleophilic phosphoryl anion. The stronger ion-pair (2.7 A) is found with DADMe-ImmH. The position of bound ImmH resembles the substrate side of the transition state barrier, and DADMe-ImmH more closely resembles the product side of the barrier. The ability to probe both substrate and product sides of the transition state barrier provides expanded opportunities to explore transition state analogue design in N-ribosyltransferases. This approach has resulted in the highest affinity transition state analogues known for MtPNP.     

Indole-3-Ethanol Oxidase in Phycomyces blakesleeanus Bgff: Characterization of the Enzyme

Indole-3-ethanol oxidase (IEt oxidase) from Phycomyces blakesleeanus Bgff.(P.b.) is a 56 kD polypeptide as determined by gel filtration. The reaction products are indole-3-acetaldehyde (IAAld) and, possibly, H₂O₂. Enzyme activity (33-45% ammonium sulfate fraction) shows a broad pH optimum and simple Michaelis-Menten kinetics (K_m 7 micromolar, Hill coefficient 0.95). Flavin adenine dinucleotide increases enzyme activity particularly under anaerobic conditions. Iodoacetate and HgCl₂ drastically inhibit the enzyme. With IAAld, product inhibition is observed at micromolar concentrations. IAA and some other acidic substituted indoles reduce enzyme activity but only at higher concentrations.     

On the Origin of Rheumatoid Arthritis: The Impact of Environment and Genes—A Population Based Twin Study

Background

Rheumatoid arthritis (RA) is an autoimmune disease with a complex origin. Previous studies have reported heritability estimates on RA at about 60%. Only 16% of the genetic background of the disease has been disclosed so far. The purpose of the present investigation was to provide an optimized estimate on the heritability of RA and to study the recurrence risk in a nationwide Caucasian twin population.

Methods and Findings

In a mail survey addressed to 56.707 twin individuals, RA was reported by 479 individuals, mean age 52 (range 16–73). Respondents underwent an interview and clinical examination. Ascertainment probability was 80%. RA was confirmed in 162 twin individuals yielding a prevalence at 0.37% (95% CI 0.31–0.43). The mean discordance time was 19 years (range 0–57). The concordance was 9.1% (95% CI 1.9 to 24.3) in MZ, 6.4% (95% CI 2.1 to 14.3) in DZss. The increased relative risk of attracting RA conditioned on having an affected cotwin compared to the background population risk was 24.6 to 35.4 in MZ twins and 17.3 to 31.6 in DZss twins. The correlation coefficients were 0.60 (0.33 to 0.78) in monozygotic (MZ) and 0.55 (0.33 to 0.72) in dizygotic same sexed (DZss) pairs. Twelve percent (95% CI 0–76%) of the phenotypic variance in the liability to RA was due to additive genetic effects, 50% (95% CI 0–72%) to shared environmental effects and 38% (95% CI 17–61%) to non-shared environmental effects.

Conclusions

This study emphasizes that family factors are important for the development of RA. Although genetic effectors are important, shared and non-shared environmental triggers and/or epigenetic stochastic events seem to be even more significant. However, it should be borne in mind that the genetic and non-genetic components may not be the same across disease subsets.     

Human immunodeficiency virus type 1 coreceptor preferences determine target T-cell depletion and cellular tropism in human lymphoid tissue

The present study sought to determine how usage of coreceptors by human immunodeficiency virus type 1 dictates cell tropism and depletion of CD4(+) T cells in human lymphoid tissues cultured ex vivo. We found that coreceptor preferences control the marked, preferential depletion of coreceptor-expressing CD4(+) lymphocytes. In addition, there was a strong, but not absolute, preference shown by CXCR4-using strains for lymphocytes and by CCR5-using strains for macrophages.     

Dissemination of Escherichia coli with CTX-M Type ESBL between Humans and Yellow-Legged Gulls in the South of France

Extended Spectrum β-Lactamase (ESBL) producing Enterobacteriaceae started to appear in the 1980s, and have since emerged as some of the most significant hospital-acquired infections with Escherichia coli and Klebsiella being main players. More than 100 different ESBL types have been described, the most widespread being the CTX-M β-lactamase enzymes (bla _CTX-M genes). This study focuses on the zoonotic dissemination of ESBL bacteria, mainly CTX-M type, in the southern coastal region of France. We found that the level of general antibiotic resistance in single randomly selected E. coli isolates from wild Yellow-legged Gulls in France was high. Nearly half the isolates (47,1%) carried resistance to one or more antibiotics (in a panel of six antibiotics), and resistance to tetracycline, ampicillin and streptomycin was most widespread. In an ESBL selective screen, 9,4% of the gulls carried ESBL producing bacteria and notably, 6% of the gulls carried bacteria harboring CTX-M-1 group of ESBL enzymes, a recently introduced and yet the most common clinical CTX-M group in France. Multi locus sequence type and phylogenetic group designations were established for the ESBL isolates, revealing that birds and humans share E. coli populations. Several ESBL producing E. coli isolated from birds were identical to or clustered with isolates with human origin. Hence, wild birds pick up E. coli of human origin, and with human resistance traits, and may accordingly also act as an environmental reservoir and melting pot of bacterial resistance with a potential to re-infect human populations.     

Influence of snag characteristics on saproxylic beetle assemblages in a south Swedish beech forest

Effects of snag characteristics on saproxylic beetles were studied in an area of managed beech forest in southern Sweden. A snag survey was combined with a beetle survey using 30 small window traps directly attached to beech snags. The total number of species was lower in the snags which were most decayed than in the three younger decay classes, while the number of red-listed species remained the same regardless of the stage of decay. The number of fresh wood species declined and the number of rot hole species increased with increasing snag decay. The diversity of fungicolous and decayed wood species peaked at the intermediate stage of decay in the snags. CCA ordination confirmed that the stage of decay in the snags was most important for species composition, followed by sun exposure. There were no general differences in species density and composition between managed and unmanaged stands. Our study suggests that most species are able to find suitable habitat within a radius of a few kilometres and that the total amount of habitat in an unfragmented forest area is more important for species diversity than the spatial distribution of this habitat. Our data also shows that species diversity increases with habitat diversity. Snags formed from giant beech trees seem to be particularly important for rare species living in rot holes. We conclude that for a high species diversity there is a requirement for snags in different stages of decay, size and degree of sun exposure.     

Temporal Variability of the Bioaerosol Background at a Subway Station: Concentration Level,Size Distribution,and Diversity of Airborne Bacteria

Naturally occurring bioaerosol environments may present a challenge to biological detection-identification-monitoring (BIODIM) systems aiming at rapid and reliable warning of bioterrorism incidents. One way to improve the operational performance of BIODIM systems is to increase our understanding of relevant bioaerosol backgrounds. Subway stations are enclosed public environments which may be regarded as potential bioterrorism targets. This study provides novel information concerning the temporal variability of the concentration level, size distribution, and diversity of airborne bacteria in a Norwegian subway station. Three different air samplers were used during a 72-h sampling campaign in February 2011. The results suggested that the airborne bacterial environment was stable between days and seasons, while the intraday variability was found to be substantial, although often following a consistent diurnal pattern. The bacterial levels ranged from not detected to 10³ CFU m⁻³ and generally showed increased levels during the daytime compared to the nighttime levels, as well as during rush hours compared to non-rush hours. The airborne bacterial levels showed rapid temporal variation (up to 270-fold) on some occasions, both consistent and inconsistent with the diurnal profile. Airborne bacterium-containing particles were distributed between different sizes for particles of >1.1 μm, although ∼50% were between 1.1 and 3.3 μm. Anthropogenic activities (mainly passengers) were demonstrated as major sources of airborne bacteria and predominantly contributed 1.1- to 3.3-μm bacterium-containing particles. Our findings contribute to the development of realistic testing and evaluation schemes for BIODIM equipment by providing information that may be used to simulate operational bioaerosol backgrounds during controlled aerosol chamber-based challenge tests with biological threat agents.     

Effects of temperature and hydrology on growth of shovelnose sturgeon Scaphirhynchus platorynchus (Rafinesque, 1820) in the lower Mississippi River

We evaluated the effects of thermal and hydrologic conditions on the growth of shovelnose sturgeon (Scaphirhynchus platorynchus) in the lower Mississippi River, USA. Duration of water temperatures 12–24°C had a positive influence and temperatures below 11°C had a negative influence on annual growth increment, but these two variables accounted for less than 15% of the variation in growth. Duration of water temperatures above 28°C, duration of floodplain inundation, duration of low water, and minimum and maximum river stage did not influence annual growth increment. Growth of shovelnose sturgeon in the lower Mississippi River appears to be positively influenced by duration of moderate water temperatures but minimally influenced by hydrologic conditions. The low variation accounted for by thermal and hydrologic variables suggests annual growth increment may be largely influenced by additional abiotic or biotic factors.     

Higher nitrate-reducer diversity in macrophyte-colonized compared to unvegetated freshwater sediment

Freshwater macrophytes stimulate rhizosphere-associated coupled nitrification–denitrification and are therefore likely to influence the community composition and abundance of rhizosphere-associated denitrifiers and nitrate reducers. Using the narG gene, which encodes the catalytic subunit of the membrane-bound nitrate reductase, as a molecular marker, the community composition and relative abundance of nitrate-reducing bacteria were compared in the rhizosphere of the freshwater macrophyte species Littorella uniflora and Myriophyllum alterniflorum to nitrate-reducing communities in unvegetated sediment. Microsensor analysis indicated a higher availability of oxygen in the rhizosphere compared to unvegetated sediment, with a stronger release of oxygen from the roots of L. uniflora compared to M. alterniflorum. Comparison of narG clone libraries between samples revealed a higher diversity of narG phylotypes in association with the macrophyte rhizospheres compared to unvegetated sediment. Quantitative PCR targeting narG- and 16S rRNA-encoding genes pointed to a selective enrichment of narG gene copies in the rhizosphere. The results suggested that the microenvironment of macrophyte rhizospheres, characterized by the release of oxygen and labile organic carbon from the root system, had a stimulating effect on the diversity and relative abundance of rhizosphere-associated nitrate reducers.