Pol32 is required for Pol zeta-dependent translesion synthesis and prevents double-strand breaks at the replication fork

POL32 encodes a non-essential subunit of Polδ and plays a role in Polδ processivity and DNA repair. In order to understand how Pol32 is involved in these processes, we performed extensive genetic analysis and demonstrated that POL32 is required for Polζ-mediated translesion synthesis, but not for Polη-mediated activity. Unlike Polζ, inactivation of Pol32 does not result in decreased spontaneous mutagenesis, nor does it limit genome instability in the absence of the error-free postreplication repair pathway. In contrast, inactivation of Pol32 results in an increased rate of replication slippage and recombination. A genome-wide synthetic lethal screen revealed that in the absence of Pol32, homologous recombination repair and cell cycle checkpoints play crucial roles in maintaining cell survival and growth. These results are consistent with a model in which Pol32 functions as a coupling factor to facilitate a switch from replication to translesion synthesis when Polδ encounters replication-blocking lesions. When Pol32 is absent, the S-phase checkpoint complex Mrc1–Tof1 becomes crucial to stabilize the stalled replication fork and recruit Top3 and Sgs1. Lack of any of the above activities will cause double strand breaks at or near the replication fork that require recombination as well as Rad9 for cell survival.     

Development of a homogeneous time-resolved fluorescence leukotriene B4 assay for determining the activity of leukotriene A4 hydrolase

Leukotriene A4 (LTA4) hydrolase catalyzes a rate-limiting final biosynthetic step of leukotriene B4 (LTB4), a potent lipid chemotactic agent and proinflammatory mediator. LTB4 has been implicated in the pathogenesis of various acute and chronic inflammatory diseases, and thus LTA4 hydrolase is regarded as an attractive therapeutic target for anti-inflammation. To facilitate identification and optimization of LTA4 hydrolase inhibitors, a specific and efficient assay to quantify LTB4 is essential. This article describes the development of a novel 384-well homogeneous time-resolved fluorescence assay for LTB4 (LTB4 HTRF assay) and its application to establish an HTRF-based LTA4 hydrolase assay for lead optimization. This LTB4 HTRF assay is based on competitive inhibition and was established by optimizing the reagent concentration, buffer composition, incubation time, and assay miniaturization. The optimized assay is sensitive, selective, and robust, with a Z' factor of 0.89 and a subnanomolar detection limit for LTB4. By coupling this LTB4 HTRF assay to the LTA4 hydrolase reaction, an HTRF-based LTA4 hydrolase assay was established and validated. Using a test set of 16 LTA4 hydrolase inhibitors, a good correlation was found between the IC50 values obtained using LTB4 HTRF with those determined using the LTB enzyme-linked immunoassay (R = 0.84). The HTRF-based LTA4 hydrolase assay was shown to be an efficient and suitable assay for determining compound potency and library screening to guide the development of potent inhibitors of LTA4 hydrolase.     

Genetic ablation of glutaredoxin-1 causes enhanced resolution of airways hyperresponsiveness and mucus metaplasia in mice with allergic airways disease

Protein-S-glutathionylation (PSSG) is an oxidative modification of reactive cysteines that has emerged as an important player in pathophysiological processes. Under physiological conditions, the thiol transferase, glutaredoxin-1 (Glrx1) catalyses deglutathionylation. Although we previously demonstrated that Glrx1 expression is increased in mice with allergic inflammation, the impact of Glrx1/PSSG in the development of allergic airways disease remains unknown. In the present study we examined the impact of genetic ablation of Glrx1 in the pathogenesis of allergic inflammation and airway hyperresponsiveness (AHR) in mice. Glrx1(-/-) or WT mice were subjected to the antigen, ovalbumin (OVA), and parameters of allergic airways disease were evaluated 48 h after three challenges, and 48 h or 7 days after six challenges with aerosolized antigen. Although no clear increases in PSSG were observed in WT mice in response to OVA, marked increases were detected in lung tissue of mice lacking Glrx1 48 h following six antigen challenges. Inflammation and expression of proinflammatory mediators were decreased in Glrx1(-/-) mice, dependent on the time of analysis. WT and Glrx1(-/-) mice demonstrated comparable increases in AHR 48 h after three or six challenges with OVA. However, 7 days postcessation of six challenges, parameters of AHR in Glrx1(-/-) mice were resolved to control levels, accompanied by marked decreases in mucus metaplasia and expression of Muc5AC and GOB5. These results demonstrate that the Glrx1/S-glutathionylation redox status in mice is a critical regulator of AHR, suggesting that avenues to increase S-glutathionylation of specific target proteins may be beneficial to attenuate AHR.     

Diagnostic utility of snail in metaplastic breast carcinoma

Aims

As one of the five Lactate dehydrogenase (LDH) isoenzymes, LDH5 has the highest efficiency to catalyze pyruvate transformation to lactate. LDH5 overexpression in cancer cells induces an upregulated glycolytic metabolism and reduced dependence on the presence of oxygen. Here we analyzed LDH5 protein expression in a well characterized large cohort of primary lung cancers in correlation to clinico-pathological data and its possible impact on patient survival.

Methods

Primary lung cancers (n = 269) and non neoplastic lung tissue (n = 35) were tested for LDH5 expression by immunohistochemistry using a polyclonal LDH5 antibody (ab53010). The results of LDH5 expression were correlated to clinico-pathological data as well as to patient's survival. In addition, the results of the previously tested Transketolase like 1 protein (TKTL1) expression were correlated to LDH5 expression.

Results

89.5% (n = 238) of NSCLC revealed LDH5 expression whereas LDH5 expression was not detected in non neoplastic lung tissues (n = 34) (p < 0.0001). LDH5 overexpression was associated with histological type (adenocarcinoma = 57%, squamous cell carcinoma = 45%, large cell carcinoma = 46%, p = 0.006). No significant correlation could be detected with regard to TNM-stage, grading or survival. A two sided correlation between the expression of TKTL1 and LDH5 could be shown (p = 0.002) within the overall cohort as well as for each grading and pN group. A significant correlation between LDH5 and TKTL1 within each histologic tumortype could not be revealed.

Conclusions

LDH5 is overexpressed in NSCLC and could hence serve as an additional marker for malignancy. Furthermore, LDH5 correlates positively with the prognostic marker TKTL1. Our results confirm a close link between the two metabolic enzymes and indicate an alteration in the glucose metabolism in the process of malignant transformation.     

Novel methoxy-carotenoids from the burgundy-colored plumage of the Pompadour Cotinga Xipholena punicea

Recent advances in the fields of chromatography, mass spectrometry, and chemical analysis have greatly improved the efficiency with which carotenoids can be extracted and analyzed from avian plumage. Prior to these technological developments, Brush (1968) [1] concluded that the burgundy-colored plumage of the male pompadour Cotinga Xipholena punicea is produced by a combination of blue structural color and red carotenoids, including astaxanthin, canthaxanthin, isozeaxanthin, and a fourth unidentified, polar carotenoid. However, X. punicea does not in fact exhibit any structural coloration. This work aims to elucidate the carotenoid pigments of the burgundy color of X. punicea plumage using advanced analytical methodology. Feathers were collected from two burgundy male specimens and from a third aberrant orange-colored specimen. Pigments were extracted using a previously published technique (McGraw et al. (2005) [2]), separated by high-performance liquid chromatography (HPLC), and analyzed by UV/Vis absorption spectroscopy, chemical analysis, mass spectrometry, nuclear magnetic resonance (NMR), and comparison with direct synthetic products. Our investigation revealed the presence of eight ketocarotenoids, including astaxanthin and canthaxanthin as reported previously by Brush (1968) [1]. Six of the ketocarotenoids contained methoxyl groups, which is rare for naturally-occurring carotenoids and a novel finding in birds. Interestingly, the carotenoid composition was the same in both the burgundy and orange feathers, indicating that feather coloration in X. punicea is determined not only by the presence of carotenoids, but also by interactions between the bound carotenoid pigments and their protein environment in the barb rami and barbules. This paper presents the first evidence of metabolically-derived methoxy-carotenoids in birds.     

Anillin and the septins promote asymmetric ingression of the cytokinetic furrow

During cytokinesis, constriction of a cortical contractile ring generates a furrow that partitions one cell into two. The contractile ring contains three filament systems: actin, bipolar myosin II filaments, and septins, GTP-binding hetero-oligomers that polymerize to form a membrane-associated lattice. The contractile ring also contains a potential filament crosslinker, Anillin, that binds all three filament types. Here, we show that the contractile ring possesses an intrinsic symmetry-breaking mechanism that promotes asymmetric furrowing. Asymmetric ingression requires Anillin and the septins, which promote the coalescence of components on one side of the contractile ring, but is insensitive to a 10-fold reduction in myosin II levels. When asymmetry is disrupted, cytokinesis becomes sensitive to partial inhibition of contractility. Thus, asymmetric furrow ingression, a prevalent but previously unexplored feature of cell division in metazoans, is generated by the action of two conserved furrow components and serves a mechanical function that makes cytokinesis robust.     

The selenium-rich C-terminal domain of mouse selenoprotein P is necessary for the supply of selenium to brain and testis but not for the maintenance of whole body selenium

Selenoprotein P (Sepp1) has two domains with respect to selenium content: the N-terminal, selenium-poor domain and the C-terminal, selenium-rich domain. To assess domain function, mice with deletion of the C-terminal domain have been produced and compared with Sepp1-/- and Sepp1+/+ mice. All mice studied were males fed a semipurified diet with defined selenium content. The Sepp1 protein in the plasma of mice with the C-terminal domain deleted was determined by mass spectrometry to terminate after serine 239 and thus was designated Sepp1Delta240-361. Plasma Sepp1 and selenium concentrations as well as glutathione peroxidase activity were determined in the three types of mice. Glutathione peroxidase and Sepp1Delta240-361 accounted for over 90% of the selenium in the plasma of Sepp1Delta240-361 mice. Calculations using results from Sepp1+/+ mice revealed that Sepp1, with a potential for containing 10 selenocysteine residues, contained an average of 5 selenium atoms per molecule, indicating that shortened and/or selenium-depleted forms of the protein were present in these wild-type mice. Sepp1Delta240-361 mice had low brain and testis selenium concentrations that were similar to those in Sepp1-/- mice but they better maintained their whole body selenium. Sepp1Delta240-361 mice had depressed fertility, even when they were fed a high selenium diet, and their spermatozoa were defective and morphologically indistinguishable from those of selenium-deficient mice. Neurological dysfunction and death occurred when Sepp1Delta240-361 mice were fed selenium-deficient diet. These phenotypes were similar to those of Sepp1-/- mice but had later onset or were less severe. The results of this study demonstrate that the C terminus of Sepp1 is critical for the maintenance of selenium in brain and testis but not for the maintenance of whole body selenium.     

Fungal endophytes of native Gossypium species in Australia

Fungal endophytes of 17 genera were found in stems of four native Gossypium species (G. australe, G. bickii, G. nelsonii, G. sturtianum) collected from inland areas in Queensland, the Northern Territory, and South Australia in 2001. Phoma, Alternaria, Fusarium, Botryosphaeria, Dichomera, and Phomopsis were common, accounting for 58, 18, 11, 3, 1, and 1 % of the 281 recovered isolates, respectively, and occurring in 47, 29, 19, 5, 5, and 4 % of the 79 sampled populations. Among the four Gossypium species in Queensland and the Northern Territory, Alternaria spp. and Fusarium spp. had the greatest recovery frequency in G. bickii stems. The recovery frequencies of Phoma spp. and Alternaria spp. were significantly greater in the G. sturtianum stems collected from South Australia than in those from Queensland and the Northern Territory. Pathogenicity of 42 representative isolates was tested on cultivated cotton (G. hirsutum). All isolates caused some localized discoloration in stem tissue when inoculation was conducted with the stem puncturing method, but none of the isolates could induce any foliar symptoms during the five-week experimental period by either inoculation method (root dipping or stem puncturing), suggesting that the endophytic fungi of native Gossypium species are unlikely sources of cotton pathogens.