Regulation of adenosine receptors expression in rat B lymphocytes by insulin

Development of diabetes is associated with altered expression of adenosine receptors (ARs). Some of these alterations might be attributed to changes in insulin concentration. This study was undertaken to investigate the possible insulin effect on ARs level, and to determine the signaling pathway utilized by insulin to regulate the expression of ARs in rat B lymphocytes. Western blot analysis of B lymphocytes protein extracts indicated that all four ARs were present at detectable levels in the cells cultured for 24 h without insulin (≤10^?11 M), although the protein band of A_2A‐AR was barely visible. Inclusion of insulin (10^?8 M) in the culture medium resulted in an increase of A₁‐AR and A_2A‐AR protein levels and a significant decrease of A_2B‐AR protein, whereas the protein level of A₃‐AR remained unchanged. Alterations in the ARs protein content were accompanied by changes in the ARs mRNA levels. Increase of the insulin concentration from 10^?11 to 10^?8 M resulted in 50% decrease of A_2B‐AR mRNA level and two‐, and threefold increase of A₁‐AR and A_2A‐AR mRNA levels, respectively. Pretreatment of B cells with cycloheximide completely blocked the insulin action on A₁‐AR and A_2A‐AR mRNA, but not on A_2B‐AR expression. Detailed pharmacological analysis demonstrated that insulin‐induced A₁‐AR and A_2A‐AR mRNA expression through the Ras/Raf‐1/MEK/ERK pathway. The insulin effect on A_2B‐AR expression was blocked by p38 MAP kinase inhibitor (SB 203580). Concluding, elevated insulin concentration differentially affects the expression of ARs in B lymphocytes in a fashion that might enhance the various immunomodulatory effects of adenosine. J. Cell. Biochem. 109: 396–405, 2010. © 2009 Wiley‐Liss, Inc.     

Offspring sex ratio skew in the sexually monomorphic house martin Delichon urbicum

Sex ratio at conception may be under selection pressure, given that male and female offspring differ in the cost of production or generate different fitness returns under specific conditions. We studied adjustments in the primary, secondary and tertiary sex ratio in house martin Delichon urbicum, which is a sexually monomorphic, socially monogamous, colonial bird. Males of this species engage in extra‐pair copulations with heavy males acquiring the highest fertilization success. We analyzed variation in the sex ratio in relation to clutch size and parental characteristics including body condition, wing length, as well as length and pigmentation of the white rump patch during three breeding seasons. The only variable which significantly explained the variation in the sex ratio was wing length of the social father and mother. The proportion of sons among offspring was positively correlated to wing length of the social father and negatively correlated to mother wing length. Social father wing length positively correlated with mean brood body mass at fledging, which may suggest that females that mated with long‐winged males produced sons, which acquired the highest total fertilization success. Consequently, our results indicate that house martin females may adaptively adjust offspring sex composition at egg laying in relation to the characteristics of their social mate.     

Influence of Pruning Measures on Recovery of Bois Noir‐infected Grapevines

In recent years, visual and analytical observations revealed a significant increase of ‘Bois noir’ (BN) in Austrian vineyards. Removing infected parts by pruning can prevent or reduce spread of the pathogen within the vines. Knowledge about the effect of pruning practices on recovery rates is essential for grapevine growers. Vines showing BN for the first time were visually categorized into classes of symptoms according to disease severity. In the ensuing winter, plants were pollarded 15 cm above the graft union (511 vines), cane pruned (529 vines) or spur pruned (heavy pruning of canes leaving spurs only; 31 vines). Pollarding resulted in significantly higher recovery rates (yearly average 62–84%) in the next growing season and significantly lower recurrence rates in the following years than cane pruning (yearly average 29–49% in the next growing season). Spur pruning was statistically indistinguishable from cane pruning. Our data allowed the conclusion that extensive removal of infected wood is crucial for immediate and persistent success of pruning measures. Recovery was significantly influenced by the severity of BN, by the cultivar and by the observation year. With pollarding treatments, a significant correlation between recovery and plant age was noticed.     

Simultaneous determination of flavonoids and phenylethanoids in the flowers of Verbascum densiflorum and V. phlomoides by high‐performance liquid chromatography

Introduction – Mullein (Verbascum) flowers are highly valued herbal drugs used in the treatment of inflammation, asthma, spasmodic coughs and other respiratory tract diseases. Their phenolic constituents are considered to be responsible for the anti‐inflammatory and antimicrobial activity of the herb. However, knowledge about the contents of phenolics in flowers is limited and no HPLC method for their analysis is available. Objective – To develop and validate an RP‐HPLC‐UV method for the simultaneous determination of eight flavonoids and two phenylethanoids in the flowers of Verbascum densiflorum and V. phlomoides. Methodology – HPLC separation was accomplished on a C₁₈ Lichrosphere 100 column (5 µm, 250 mm × 4.6 mm, i.d.) with an acetonitrile gradient elution using aqueous 0.5% (w/v) orthophosphoric acid solution containing 1% (v/v) tetrahydrofurane. Results – All the calibration curves showed good linear correlation coefficients (r > 0.997) over the wide test ranges. The relative standard deviation of the method was less than 3.4% for intra‐ and inter‐day assays, and the average recoveries were between 93.5 and 101.9%. High sensitivity was demonstrated with detection limits of 0.062–0.083 µg/mL for flavonoid aglycones, 0.156–0.336 µg/mL for flavonoid glycosides and 0.390–0.555 µg/mL for phenylethanoids. The flower samples of V. phlomoides were found to contain high levels of diosmin and tamarixetin 7‐rutinoside (2.327–2.392% of dry weight), whereas verbascoside (0.688–0.742% of dry weight) and luteolin 7‐glucoside (0.204–0.279% of dry weight) dominated in the V. densiflorum flower. Conclusion – The HPLC method established is appropriate for the quality assurance and the differentiation of V. phlomoides and V. densiflorum samples. Copyright © 2009 John Wiley & Sons, Ltd.     

Quantification of the emetic toxin cereulide in food products by liquid chromatography-mass spectrometry using synthetic cereulide as a standard

Bacillus cereus produces the emetic toxin cereulide, a cyclic dodecadepsipeptide that can act as a K(+) ionophore, dissipating the transmembrane potential in mitochondria of eukaryotic cells. Because pure cereulide has not been commercially available, cereulide content in food samples has been expressed in valinomycin equivalents, a highly similar cyclic potassium ionophore that is commercially available. This research tested the biological activity of synthetic cereulide and validated its use as a standard in the quantification of cereulide contents in food samples. The synthesis route consists of 10 steps that result in a high yield of synthetic cereulide that showed biological activity in the HEp-2 cell assay and the boar sperm motility assay. The activity is different in both methods, which may be attributed to differences in K(+) content of the test media used. Using cereulide or valinomycin as a standard to quantify cereulide based on liquid chromatography-mass spectrometry (LC-MS), the concentration determined with cereulide as a standard was on average 89.9% of the concentration determined using valinomycin as a standard. The recovery experiments using cereulide-spiked food products and acetonitrile as extraction solute showed that the LC-MS method with cereulide as a standard is a reliable and accurate method to quantify cereulide in food, because the recovery rate was close to 100% over a wide concentration range.     

Mutation analysis of mitochondrial 12S rRNA gene in Polish patients with non-syndromic and aminoglycoside-induced hearing loss

Mutations in mitochondrial DNA have been reported as associated with non-syndromic and aminoglycoside-induced hearing loss. In the present study, we have performed mutational screening of entire 12S rRNA gene in 250 unrelated patients with non-syndromic and aminoglycoside-induced hearing loss. Twenty-one different homoplasmic sequence variants were identified, including eight common polymorphisms, one deafness-associated mutation m.1555 A>G and three putatively pathogenic variants: m.669 T>C, m.827 A>G, m.961 delT+C(n)ins. The incidence of m.1555 A>G was estimated for 3.6% (9/250); however, where aminoglycoside exposure was taken as a risk factor, the frequency was 5.5% (7/128). Substitution m.669 T>C was identified only in patients with hearing impairment and episode of aminoglycoside exposure, which may suggest that such additional risk factors must appear to induce clinical phenotype. Moreover, two 12S rRNA sequence variants: m.988 G>A and m.1453 A>G, localized at conserved sites and affected RNA secondary structure, may be new candidates for non-syndromic and aminoglycoside-induced hearing loss associated mutations.     

Multiple ASF/SF2 Sites in the Human Papillomavirus Type 16 (HPV-16) E4-Coding Region Promote Splicing to the Most Commonly Used 3′-Splice Site on the HPV-16 Genome

Our results presented here demonstrate that the most abundant human papillomavirus type 16 (HPV-16) mRNAs expressing the viral oncogenes E6 and E7 are regulated by cellular ASF/SF2, itself defined as a proto-oncogene and overexpressed in cervical cancer cells. We show that the most frequently used 3′-splice site on the HPV-16 genome, site SA3358, which is used to produce primarily E4, E6, and E7 mRNAs, is regulated by ASF/SF2. Splice site SA3358 is immediately followed by 15 potential binding sites for the splicing factor ASF/SF2. Recombinant ASF/SF2 binds to the cluster of ASF/SF2 sites. Mutational inactivation of all 15 sites abolished splicing to SA3358 and redirected splicing to the downstream-located, late 3′-splice site SA5639. Overexpression of a mutant ASF/SF2 protein that lacks the RS domain, also totally inhibited the usage of SA3358 and redirected splicing to the late 3′-splice site SA5639. The 15 ASF/SF2 binding sites could be replaced by an ASF/SF2-dependent, HIV-1-derived splicing enhancer named GAR. This enhancer was also inhibited by the mutant ASF/SF2 protein that lacks the RS domain. Finally, silencer RNA (siRNA)-mediated knockdown of ASF/SF2 caused a reduction in spliced HPV-16 mRNA levels. Taken together, our results demonstrate that the major HPV-16 3′-splice site SA3358 is dependent on ASF/SF2. SA3358 is used by the most abundantly expressed HPV-16 mRNAs, including those encoding E6 and E7. High levels of ASF/SF2 may therefore be a requirement for progression to cervical cancer. This is supported by our earlier findings that ASF/SF2 is overexpressed in high-grade cervical lesions and cervical cancer.Human papillomavirus type 16 (HPV-16) is the foremost cause of cervical cancer, which is one of the most common cancers in women globally (10, 37). Persistence of high-risk HPV types, such as HPV-16, is the highest risk factor for the development of cervical cancer. The majority of all DNA viruses that establish persistence have evolved a highly organized gene expression program, often divided into clear early and late phases. The HPV-16 genome contains an early promoter that could potentially express mRNAs encoding all viral gene products, and a late differentiation-dependent promoter that specifically excludes expression of E6 and E7 (21). The switch from early to late gene expression includes a promoter switch as well as derepression and activation of the late poly(A) signal and late splice sites (16). To activate late splice sites and the late poly(A) signal, many early splice sites and the early poly(A) signal must be downregulated to allow for competition from mutually exclusive late splice sites and poly(A) signal (8, 26, 36). Other HPV-16 splice sites are used by both early and late mRNAs and should function well in both mitotic cells and terminally differentiated cells. One of the major splice sites used by both early and late mRNAs is SA3358 (Fig. ​(Fig.1A).1A). This splice site is outstanding in that it is used to produce the majority of all HPV-16 mRNAs, including the mRNAs of the oncogenes E6 and E7 and the E4, E5, L1, and perhaps L2 proteins. In contrast, efficient usage of SA3358 specifically prevents expression of HPV-16 E1 and E2.Open in a separate windowFIG. 1.(A) Schematic representation of the HPV-16 genome. Early and late viral promoters p97 and p670 are indicated. Numbers indicate nucleotide positions of 5′-splice sites (filled circles), 3′-splice sites (open circles), or early and late poly(A) signals pAE and pAL, respectively. LCR, long control region. A few selected early and late mRNAs are shown (1). Previously described splicing silencers and enhancers are indicated (24, 34, 35). (B) Diagram with potential ASF/SF2 sites upstream and downstream of SD3632 predicted by ESEfinder (4). Heights of the bars represent degrees of similarity to ASF/SF2 binding sites according to ESEfinder. HPV-16 splice sites SA3358 and SD3632 are indicated. Numbers indicate nucleotide positions in the HPV-16 genome. The position of a previously described enhancer is indicated (24). (C) ASF/SF2 sites in the mutant HPV-16 sequence in which the ASF/SF2 sites had been inactivated, as predicted by ESEfinder (4). (D) Exact sequences of the wt and mutant (mut) HPV-16 Predicted sequences between nucleotide positions 3407 and 3627 in the HPV-16 genome. Dots represent identical nucleotides.Many, if not all, HPV types contain a 3′-splice site in the E4 open reading frame (orf) that is spliced to an upstream 5′-splice site that joins the E1 AUG with the E4 orf. In HPV-16, these splice sites are named SA3358 and SD880 (Fig. ​(Fig.1A),1A), whereas they are named SD847 and SA3325 in HPV-11 and SD877 and SA3295 in HPV-31 (1). Splicing between HPV-16 SD880 and SA3358 (6, 9, 27), or the corresponding sites in HPV-11 (5, 20, 23) and HPV-31 (11, 12), occurs on the most-common early mRNAs encoding E6 and E7, as well as on the most-abundant late mRNA encoding E4. In addition, the most-common L1 mRNA is also spliced between SD880 and SA3358 (17), or the corresponding sites in HPV-11 (23) and HPV-31 (12, 22). Analysis of HPV-16 splicing in cervical scrape samples revealed that splicing between SD880 and SA3358 was the most-common splicing event in both low- and high-grade cervical lesions (25). In vitro transfection experiments demonstrated that splicing to SA3358 was required for efficient expression of E6 and E7 (2). As a matter of fact, splicing between SD880 and SA3358 was required for production of E6 and E7 quantities that were needed for transformation of cells by these HPV proteins. In HPV-31, SA3295 corresponds to HPV-16 SA3358. Mutational inactivation of HPV-31 SA3295 in an infectious molecular clone of HPV-31 immediately caused splicing to a cryptic 3′-splice site located three nucleotides further down (15). These results indicated that HPV-31 SA3295 is under the control of strong splicing enhancer elements and that there is a strong pressure on the virus to maintain a 3′-splice site in that exact region.We have previously reported that HPV-16 SA3358 has an exceptionally poor 3′-splice site sequence compared to a consensus 3′-splice site (24). This is due primarily to an almost complete absence of an upstream row of uninterrupted pyrimidines that normally characterize an efficiently utilized 3′-splice site. However, SA3358 is one of the most efficiently used splice sites on the HPV-16 genome (24, 33). We have previously shown that utilization of HPV-16 SA3358 is totally dependent on exonic sequences downstream of SA3358, and we concluded that a splicing enhancer was located downstream of SA3358 (24). Here, we have followed up these findings; we demonstrate that the enhancer elements downstream of HPV-16 SA3358 are binding sites for ASF/SF2, and we show that ASF/SF2 enhances splicing to SA3358.     

DARPin-Assisted Crystallography of the CC2-LZ Domain of NEMO Reveals a Coupling between Dimerization and Ubiquitin Binding

NEMO is an integral part of the IκB kinase complex and serves as a molecular switch by which the NF-κB signaling pathway can be regulated. Oligomerization and polyubiquitin (poly-Ub) binding, mediated through the regulatory CC2-LZ domain, were shown to be key features governing NEMO function, but the relationship between these two activities remains unclear. In this study, we solved the structure of this domain in complex with a designed ankyrin repeat protein, which helps its crystallization. We generated several NEMO mutants in this domain, including those associated with human diseases incontinentia pigmenti and immunodeficiency with or without anhidrotic ectodermal dysplasia. Analytical ultracentrifugation and thermal denaturation experiments were used to evaluate the dimerization properties of these mutants. A fluorescence-based assay was developed, as well, to quantify the interaction to monoubiquitin and poly-Ub chains. Moreover, the effect of these mutations was investigated for the full-length protein. We show that a proper folding of the ubiquitin-binding domain, termed NOA/UBAN/NUB, into a stable coiled-coil dimer is required but not sufficient for efficient interaction with poly-Ub. In addition, we show that binding to poly-Ub and, to a lesser extent, to monoubiquitin increases the stability of the NOA coiled-coil dimer. Collectively, these data provide structural insights into how several pathological mutations within and outside of the CC2-LZ's NOA ubiquitin binding site affect IκB kinase activation in the NF-κB signaling pathway.