Structure of a Bimodular Botulinum Neurotoxin Complex Provides Insights into Its Oral Toxicity

Botulinum neurotoxins (BoNTs) are produced by Clostridium botulinum and cause the fatal disease botulism, a flaccid paralysis of the muscle. BoNTs are released together with several auxiliary proteins as progenitor toxin complexes (PTCs) to become highly potent oral poisons. Here, we report the structure of a ∼760 kDa 14-subunit large PTC of serotype A (L-PTC/A) and reveal insight into its absorption mechanism. Using a combination of X-ray crystallography, electron microscopy, and functional studies, we found that L-PTC/A consists of two structurally and functionally independent sub-complexes. A hetero-dimeric 290 kDa complex protects BoNT, while a hetero-dodecameric 470 kDa complex facilitates its absorption in the harsh environment of the gastrointestinal tract. BoNT absorption is mediated by nine glycan-binding sites on the dodecameric sub-complex that forms multivalent interactions with carbohydrate receptors on intestinal epithelial cells. We identified monosaccharides that blocked oral BoNT intoxication in mice, which suggests a new strategy for the development of preventive countermeasures for BoNTs based on carbohydrate receptor mimicry.     

Essential oils from buds and leaves of two hazelnut (Corylus L.) cultivars with different resistance to filbert big bud mite (Phytoptus avellanae Nal.) and filbert aphid (Myzocallis coryli Goetze)

The aim of this study was to identify the allelopatic effect of the components of a mixture of essential oils (EO) contained in the buds and leaves of hazel (Corylus L.) on herbivores. We examined the effect of these compounds on the choice of plants of two different hazel cultivars by Phytoptus avellanae Nal. (filbert big bud mite) and Myzocallis coryli Goetze (filbert aphid), which are the most important pests of hazel in Poland and throughout the world. Our results show that plants of cv. ‘Mogulnus’ were more resistant than those of cv. ‘Barra’ to the feeding of mites and aphids in all study years. Using gas chromatography (GC) and GC/mass spectrometry methodology, we determined the qualitative and quantitative composition of EO in the buds and leaves of plants of these two hazel cultivars. The EO obtained from the analysed materials was a mixture of mono- and sesquiterpenes. The emission of volatile organic compounds from plants is known to repel or attract pests. The mixture of EO present in the hazel buds of cv. ‘Mogulus’, which is resistant to filbert big bud mite, was characterized by a high content of nerol, α-campholenol, methyl salicylate, spatulenol, β-caryophylene and δ-cadinene. In contrast, the leaves of this cultivar, colonized by filbert aphid but to a relatively small extent, contained greater quantities of nerol, α-campholenol, p-cymene, α-terpineol and germacrene D, than the leaves of cv. ‘Barra’, which is more accepted by aphids. However, the leaves of cv. ‘Barra’ were characterized by a considerably high content of menthol, limonene, isomenthone, methyl salicylate and L-menthone.     

Genetic Polymorphism of Human Y Chromosome and Risk Factors for Cardiovascular Diseases: A Study in WOBASZ Cohort

Genetic variants of Y chromosome predispose to hypertension in rodents, whereas in humans the evidence is conflicting. Our purpose was to study the distribution of a panel of Y chromosome markers in a cohort from a cross-sectional population-based study on the prevalence of cardiovascular risk factors in Poland (WOBASZ study). The HindIII, YAP Y chromosome variants, previously shown to influence blood pressure, lipid traits or height, as well as SNPs defining main Y chromosome haplogroups, were typed in 3026, 2783 and 2652 samples, respectively. In addition, 4 subgroups (N∼100 each) representing extremes of LDL concentration or blood pressure (BP) were typed for a panel of 17 STRs. The HindIII and YAP polymorphism were not associated with any of the studied traits. Analysis of the haplogroup distribution showed an association between higher HDL level and hg I-M170 (P = 0.02), higher LDL level and hg F*(xI-M170, J2-M172, K-M9) (P = 0.03) and lower BMI and hg N3-Tat (P = 0.04). Analysis of STRs did not show statistically significant differences. Since all these associations lost statistical significance after Bonferroni correction, we conclude that a major role of Y chromosome genetic variation (defined by HindIII, YAP or main Y chromosome haplogroups) in determining cardiovascular risk in Poles is unlikely.     

Critical Role of a Ferritin-Like Protein in the Control of Listeria monocytogenes Cell Envelope Structure and Stability under β-lactam Pressure

The human pathogen Listeria monocytogenes is susceptible to the β-lactam antibiotics penicillin G and ampicillin, and these are the drugs of choice for the treatment of listerial infections. However, these antibiotics exert only a bacteriostatic effect on this bacterium and consequently, L. monocytogenes is regarded as β-lactam tolerant. It is widely accepted that the phenomenon of bacterial tolerance to β-lactams is due to the lack of adequate autolysin activity, but the mechanisms of L. monocytogenes tolerance to this class of antibiotics are poorly characterized. A ferritin-like protein (Fri) was recently identified as a mediator of β-lactam tolerance in L. monocytogenes, but its function in this process remains unknown. The present study was undertaken to improve our understanding of L. monocytogenes tolerance to β-lactams and to characterize the role of Fri in this phenomenon. A comparative physiological analysis of wild-type L. monocytogenes and a fri deletion mutant provided evidence of a multilevel mechanism controlling autolysin activity in cells grown under β-lactam pressure, which leads to a reduction in the level and/or activity of cell wall-associated autolysins. This is accompanied by increases in the amount of teichoic acids, cell wall thickness and cell envelope integrity of L. monocytogenes grown in the presence of penicillin G, and provides the basis for the innate β-lactam tolerance of this bacterium. Furthermore, this study revealed the inability of the L. monocytogenes Δ fri mutant to deplete autolysins from the cell wall, to adjust the content of teichoic acids and to maintain their D-alanylation at the correct level when treated with penicillin G, thus providing further evidence that Fri is involved in the control of L. monocytogenes cell envelope structure and stability under β-lactam pressure.     

Effect of Cadmium on the Level of Isoprenoid-Derived Phytohormones in Duckweed Wolffia arrhiza

Journal of Plant Growth Regulation - Wolffia arrhiza (L.) Horkel ex Wimm. is an aquatic plant belonging to the Lemnaceae family. It does not have leaves, stems, and roots, flowers rarely occur,...     

Liposomal temocene (m-THPPo) photodynamic treatment induces cell death by mitochondria-independent apoptosis

Background

The cell death pathway activated after photodynamic therapy (PDT) is controlled by a variety of parameters including the chemical structure of the photosensitizer, its subcellular localization, and the photodynamic damage induced. The present study aims to characterize a suitable m-THPPo liposomal formulation, to determine its subcellular localization in HeLa cells and to establish the cell death mechanisms that are activated after photodynamic treatments.

Methods

Liposomes containing m-THPPo were prepared from a mixture of DPPC and DMPG at a 9:1 molar ratio. In order to procure the best encapsulation efficiency, the m-THPPo/lipid molar ratio was considered. HeLa cells were incubated with liposomal m-THPPo and the subcellular localization of m-THPPo was studied. Several assays such as TUNEL, annexin V/propidium iodide and Hoechst-33258 staining were performed after photodynamic treatments. The apoptotic initiation was assessed by cytochrome c and caspase-2 immunofluorescence.

Results

m-THPPo encapsulated in liposomes showed a decrease of the fluorescence and singlet oxygen quantum yields, compared to those of m-THPPo dissolved in tetrahydrofuran. Liposomal m-THPPo showed colocalization with LysoTracker® and it induced photoinactivation of HeLa cells by an apoptotic mechanism. In apoptotic cells no relocalization of cytochrome c could be detected, but caspase-2 was positive immediately after photosensitizing treatments.

Conclusions

Photodynamic treatment with liposomal m-THPPo leads to a significant percentage of apoptotic morphology of HeLa cells. The activation of caspase-2, without the relocalization of cytochrome c, indicates a mitochondrial-independent apoptotic mechanism.

General significance

These results provide a better understanding of the cell death mechanism induced after liposomal m-THPPo photodynamic treatment.     

Heterogeneity of 11q13 region rearrangements in laryngeal squamous cell carcinoma analyzed by microarray platforms and fluorescence in situ hybridization

We reinvestigated rearrangements occurring in region q13 of chromosome 11 aiming to: (i) describe heterogeneity of the observed structural alterations, (ii) estimate amplicon size and (iii) identify of oncogenes involved in laryngeal cancer progression as potential targets for therapy. The study included 17 cell lines derived from laryngeal cancers and 34 specimens from primary laryngeal tumors. The region 11q13 was analyzed by fluorescence in situ hybridization (FISH), array comparative genomic hybridization (aCGH) and gene expression microarray. Next, quantitative real time PCR was used for chosen genes to confirm results from aCGH and gene expression microarray. The observed pattern of aberrations allows to distinguish three ways, in which gain and amplification involving 11q13 region may occur: formation of a homogeneously staining region; breakpoints in/near 11q13, which lead to the three to sevenfold increase of the copy number of 11q13 region; the presence of additional copies of the whole chromosome 11. The minimal altered region of gain and/or amplification was limited to ~1.8 Mb (chr.11:69,395,184–71,209,568) and comprised mostly 11q13.3 band which contain 12 genes. Five, out of these genes (CCND1, ORAOV1, FADD, PPFIA1, CTTN) had higher expression levels in comparison to healthy controls. Apart from CCND1 gene, which has an established role in pathogenesis of head and neck cancers, CTTN, ORAOV1 and FADD genes appear to be oncogene-candidates in laryngeal cancers, while a function of PPFIA1 requires further studies.     

Changes in plasma thiol levels induced by different phases of treatment in breast cancer; the role of commercial extract from black chokeberry

Different low-molecular-weight thiols, including glutathione, cysteine, and cysteinylglycine are physiological free radical scavengers. On the other hand, homocysteine may play a role as an oxidant. The aim of our present study was to establish in vitro the effects of the commercial extract of Aronia melanocarpa (Aronox^?) on the amount of selected low-molecular-weight thiols and the activity of antioxidative enzymes (superoxide dismutase, glutathione peroxidase, and glutathione reductase) in plasma obtained from patients with invasive breast cancer during different phases of treatment [before or after the surgery and patients after different phases of chemotherapy (doxorubicin and cyclophosphamide)] and from healthy subjects. Patients were hospitalized in Department of Oncological Surgery and Department of Chemotherapy, Medical University of Lodz, Poland. The level of low-molecular-weight thiols was determined by high-performance liquid chromatography. We observed that in the presence of the Aronia extract changes in amount of thiols in plasma from breast cancer patients (at all tested groups) were significantly reduced. Our results showed that tested commercial extract reduced modifications of antioxidative enzymes activity in plasma from patients during different phases of treatment, but this effect was not statistical significant. Our results suggest that the Aronia extract supplementation in breast cancer patients has a beneficial effect on thiols concentration in plasma. Plasma, as reported in this work, could be used as an experimental model to evaluate the beneficial action of plant supplements, including phenolic extracts on thiols or other molecules during different phases of treatment.