Nitrosothiol Formation and Protection against Fenton Chemistry by Nitric Oxide-induced Dinitrosyliron Complex Formation from Anoxia-initiated Cellular Chelatable Iron Increase

Dinitrosyliron complexes (DNIC) have been found in a variety of pathological settings associated with ^•NO. However, the iron source of cellular DNIC is unknown. Previous studies on this question using prolonged ^•NO exposure could be misleading due to the movement of intracellular iron among different sources. We here report that brief ^•NO exposure results in only barely detectable DNIC, but levels increase dramatically after 1–2 h of anoxia. This increase is similar quantitatively and temporally with increases in the chelatable iron, and brief ^•NO treatment prevents detection of this anoxia-induced increased chelatable iron by deferoxamine. DNIC formation is so rapid that it is limited by the availability of ^•NO and chelatable iron. We utilize this ability to selectively manipulate cellular chelatable iron levels and provide evidence for two cellular functions of endogenous DNIC formation, protection against anoxia-induced reactive oxygen chemistry from the Fenton reaction and formation by transnitrosation of protein nitrosothiols (RSNO). The levels of RSNO under these high chelatable iron levels are comparable with DNIC levels and suggest that under these conditions, both DNIC and RSNO are the most abundant cellular adducts of ^•NO.     

Heterologous expression of an endoglucanase gene (endA) from the ruminai anaerobe Ruminococcus flavefaciens 17 in Streptococcus bovis and Streptococcus sanguis

Abstract The heterologous expression of a cloned endoglucanase gene ( endA ) from the ruminai bacterium Ruminococcus flavefaciens 17 was demonstrated in the Streptococcus species S. bovis JB1 and S. sanguis DLL The endA gene was introduced into S. bovis and S. sanguis using the Escherichia coli/Streptococcus shuttle vector pVA838. Expression of the gene was detected by clearing zones around the recombinant colonies on agar plates containing carboxymethylcellulose stained with Congo red. S. bovis JB1 containing the endA gene was capable of utilizing cellotetraose at a faster rate than the parent strain. This is the first demonstration that Streptococcus species can express a gene from a Ruminococcus flavefaciens strain.     

Formation of propionate and butyrate by the human colonic microbiota

The human gut microbiota ferments dietary non‐digestible carbohydrates into short‐chain fatty acids (SCFA). These microbial products are utilized by the host and propionate and butyrate in particular exert a range of health‐promoting functions. Here an overview of the metabolic pathways utilized by gut microbes to produce these two SCFA from dietary carbohydrates and from amino acids resulting from protein breakdown is provided. This overview emphasizes the important role played by cross‐feeding of intermediary metabolites (in particular lactate, succinate and 1,2‐propanediol) between different gut bacteria. The ecophysiology, including growth requirements and responses to environmental factors, of major propionate and butyrate producing bacteria are discussed in relation to dietary modulation of these metabolites. A detailed understanding of SCFA metabolism by the gut microbiota is necessary to underpin effective strategies to optimize SCFA supply to the host.     

Occurrence of 5-methoxypodophyllotoxin in plants,cell cultures and regenerated plants of Linum flavum

The 5-methoxypodophyllotoxin contents of plants, cell cultures and regenerated plants of Linum flavum are compared. It is demonstrated that cell cultures are able to produce amounts of 5-methoxypodophyllotoxin that are comparable to the concentration in fully differentiated plants. The production of 5-methoxy-podophyllotoxin depends on the hormonal balance of the growth medium. The use of 2,4-dichlorophenoxyacetic acid as the growth regulator is favourable for 5-methoxypodophyllotoxin production when compared to naphthylacetic acid. The 5-methoxypodophyllotoxin accumulation appears to be positively related to the internal cell volume.     

Plants increase silicon content as a response to nitrogen or phosphorus limitation: a case study with Holcus lanatus

Plant and Soil - Silicon (Si) has been shown to beneficially affect plant performance under stressful environmental conditions, such as water or nutrient deficiency. Here we tested the effects of...     

Ovarian structure and oogenesis of the oviparous goodeids Crenichthys baileyi (Gilbert, 1893) and Empetrichthys latos Miller, 1948 (teleostei,Cyprinodontiformes)

The cyprinodontiform family Goodeidae comprises two biogeographically disjunct subfamilies: the viviparous Goodeinae endemic to the Mexican Plateau, and the oviparous Empetrichthyinae, known only from relict taxa in Nevada and California. Ovarian characteristics of two oviparous species of goodeid, Crenichthys baileyi and Empetrichthys latos, studied using museum collections, are compared with those of viviparous species of goodeids. Both subfamilies have a single, cystovarian ovary. The ovary in the viviparous Goodeinae has an internal septum that divides the ovarian lumen into two compartments, and it may possess oogonia. There is no ovarian septum in the oviparous C. baileyi and E. latos. Oogenesis is similar in both subfamilies with regard to the proliferation of oogonia, initiation of meiosis, primary growth and development of an oocyte during secondary growth in which fluid yolk progressively fuses into a single globule. Notably, eggs of C. baileyi and E. latos are approximately double the size of those of the viviparous Goodeinae in which embryos develop inside the ovarian lumen and are nourished, in part, by nutrients transferred from the maternal tissues, a mode of embryo development called matrotrophy. Egg envelopes of the two subfamilies differ in that those of C. baileyi and E. latos have a relatively thick zona pellucida, attachment fibrils or filaments that develop between the follicle cells during oogenesis, and a micropyle observed only in E. latos. In contrast, viviparous goodeid eggs have a relatively thin zona pellucida, but lack adhesive fibrils, and a micropyle was not observed. These reproductive characters are compared with those of species of the eastern North American Fundulus, a representative oviparous cyprinodontiform. One newlyrecognized shared, derived character, a single, median ovoid ovary with no obvious external evidence of fusion, supports monophyly of the Goodeidae. Differences among the goodeid subfamilies and Fundulus are interpreted relative to the oviparous versus viviparous modes of reproduction. J. Morphol., 2012. © 2011 Wiley Periodicals, Inc.     

The influence of water depth and flow regime on phytoplankton biomass and community structure in a shallow,lowland river

Hydrobiologia - The taxonomic composition and biomass of phytoplankton in the San Joaquin River, California, were examined in relation to water depth, flow regime, and water chemistry. Without...     

In Vitro Intracellular Trafficking of Virulence Antigen during Infection by Yersinia pestis

Yersinia pestis, the causative agent of plague, encodes several essential virulence factors on a 70 kb plasmid, including the Yersinia outer proteins (Yops) and a multifunctional virulence antigen (V). V is uniquely able to inhibit the host immune response; aid in the expression, secretion, and injection of the cytotoxic Yops via a type III secretion system (T3SS)-dependent mechanism; be secreted extracellularly; and enter the host cell by a T3SS-independent mechanism, where its activity is unknown. To elucidate the intracellular trafficking and target(s) of V, time-course experiments were performed with macrophages (MΦs) infected with Y. pestis or Y. pseudotuberculosis at intervals from 5 min to 6 h. The trafficking pattern was discerned from results of parallel microscopy, immunoblotting, and flow cytometry experiments. The MΦs were incubated with fluorescent or gold conjugated primary or secondary anti-V (antibodies [Abs]) in conjunction with organelle-associated Abs or dyes. The samples were observed for co-localization by immuno-fluorescence and electron microscopy. For fractionation studies, uninfected and infected MΦs were lysed and subjected to density gradient centrifugation coupled with immunoblotting with Abs to V or to organelles. Samples were also analyzed by flow cytometry after lysis and dual-staining with anti-V and anti-organelle Abs. Our findings indicate a co-localization of V with (1) endosomal proteins between 10–45 min of infection, (2) lysosomal protein(s) between 1–2 h of infection, (3) mitochondrial proteins between 2.5–3 h infection, and (4) Golgi protein(s) between 4–6 h of infection. Further studies are being performed to determine the specific intracellular interactions and role in pathogenesis of intracellularly localized V.     

Genome-wide methylation profiling identifies hypermethylated biomarkers in high-grade cervical intraepithelial neoplasia

Epigenetic modifications, such as aberrant DNA promoter methylation, are frequently observed in cervical cancer. Identification of hypermethylated regions allowing discrimination between normal cervical epithelium and high-grade cervical intraepithelial neoplasia (CIN2/3), or worse, may improve current cervical cancer population-based screening programs. In this study, the DNA methylome of high-grade CIN lesions was studied using genome-wide DNA methylation screening to identify potential biomarkers for early diagnosis of cervical neoplasia. Methylated DNA Immunoprecipitation (MeDIP) combined with DNA microarray was used to compare DNA methylation profiles of epithelial cells derived from high-grade CIN lesions with normal cervical epithelium. Hypermethylated differentially methylated regions (DMRs) were identified. Validation of nine selected DMRs using BSP and MSP in cervical tissue revealed methylation in 63.2–94.7% high-grade CIN and in 59.3–100% cervical carcinomas. QMSP for the two most significant high-grade CIN-specific methylation markers was conducted exploring test performance in a large series of cervical scrapings. Frequency and relative level of methylation were significantly different between normal and cancer samples. Clinical validation of both markers in cervical scrapings from patients with an abnormal cervical smear confirmed that frequency and relative level of methylation were related with increasing severity of the underlying CIN lesion and that ROC analysis was discriminative. These markers represent the COL25A1 and KATNAL2 and their observed increased methylation upon progression could intimate the regulatory role in carcinogenesis. In conclusion, our newly identified hypermethylated DMRs represent specific DNA methylation patterns in high-grade CIN lesions and are candidate biomarkers for early detection.