The early dendritic cell signaling induced by virulent Francisella tularensis strain occurs in phases and involves the activation of ERKs and p38 in the later stage

Dendritic cells (DCs) infected by Francisella tularensis are poorly activated and do not undergo classical maturation process. While reasons of such unresponsiveness are not fully understood, their impact on the priming of immunity is well appreciated. Previous attempts to explain the behavior of Francisella-infected DCs were hypothesis-driven and focused on events at later stages of infection. Here, we took an alternative unbiased approach by applying methods of global phosphoproteomics to analyze the dynamics of cell signaling in primary DCs during the first hour of infection by Francisella tularensis. Presented results show that the early response of DCs to Francisella occurs in phases and that ERK and p38 signaling modules induced at the later stage are differentially regulated by virulent and attenuated ΔdsbA strain. These findings imply that the temporal orchestration of host pro-inflammatory pathways represents the integral part of Francisella life-cycle inside hijacked DCs

Elektrokemijsko određivanje metalotioneina kod domaće peradi

Metallothionein (MT) belongs to group of intracellular, low-molecular and cysteine-rich proteins with a molecular weight from 6 to 10 kDa. Owing to their high affinity to heavy metals (Zn, Cd, As, etc.) their main role is homeostatic control and detoxification of metal ions in an organism. In the present work we aimed at suggesting and utilizing electroanalytical techniques to determine content of MT in the blood serum of domestic fowls. Electrochemical measurements were performed with an AUTOLAB Analyser connected to VA-Stand 663, using a standard cell with three electrodes. Particularly, MT was detected by adsorptive transfer stripping technique in connection with differential pulse voltammetry. The detection limit of MT was estimated down to 100 fM (standards only) or down to 100 pM measured in the presence of blood serum. The average content of MT was 21.3 µM. The MT level in hens was about 25 % higher than in cocks. This phenomenon can be related to higher demands on the content of this protein in hens due the requirement for ion transport to form eggshell.Metalotionein (MT) pripada grupi intracelularnih proteina male molekularne mase bogatih cisteinom, s molekularnom masom od 6 do 10 kDa. Zbog njihovog afiniteta prema teškim metalima (Zn, Cd, As, itd.) njihova glavna uloga je homeostatska kontrola i detoksifikacija iona metala u organizmu. U ovom radu predlažu se elektroanalitičke tehnike za određivanje sadržaja MT u krvnom serumu domaće peradi. Elektrokemijska mjerenja izvršena su uređajem AUTOLAB Analyser povezanim s VA-Stand 663, koristeći standardnu ćeliju s tri elektrode. Osim toga MT je određivan tehnikom adsorptivnog transfera, povezanoj s voltmetrijom diferencijalnog pulsa. Granica detekcije MT je procjenjivana do 100 fM (samo standardi) ili do 100 pM, mjereno u prisutnosti krvnog seruma. Prosječni sadržaj MT bio je 21.3 µM. Razina MT kod kokoši bila je otprilike 25% viša nego kod pijetlova. Ta pojava može se objasniti većom potrebom za ovim proteinom kod kokoši zbog transporta iona prilikom stvaranja ljuske jajeta

An Adsorptive Transfer Technique Coupled with Brdicka Reaction to Reveal the Importance of Metallothionein in Chemotherapy with Platinum Based Cytostatics

The drugs based on platinum metals represent one of the oldest, but also one of the most effective groups of chemotherapeutic agents. Thanks to many clinical studies it is known that resistance of tumor cells to drugs is a frequent cause of chemotherapy failure. With regard to platinum based drugs, multidrug resistance can also be connected with increased expression of low-molecular weight protein metallothionein (MT). This study aimed at investigating the interactions of MT with cisplatin or carboplatin, using the adsorptive transfer technique coupled with differential pulse voltammetry Brdicka reaction (AdTS DPV Brdicka reaction), and a comparison of in vitro results with results obtained in vivo. The results obtained from the in vitro study show a strong affinity between platinum based drugs and MT. Further, we analyzed extracts of neuroblastoma cell lines treated with cisplatin or carboplatin. It is clear that neuroblastoma UKF-NB-4 cisplatin-resistant and cisplatin-sensitive cell lines unlikely respond to the presence of the platinum-based cytostatics cisplatin and carboplatin. Finally, we determined the level of MT in samples from rabbits treated with carboplatin and patients with retinoblastoma treated with the same drug

Altered ceramide metabolism is a feature in the extracellular vesicle-mediated spread of alpha-synuclein in Lewy body disorders

This study was funded by the Lewy Body Society and the Michael J. Fox Foundation for Parkinson’s Research. A.H. and B.M.B. gratefully acknowledge funding from the Knut och Alice Wallenberg Stiftelse through the Wallenberg Centre for Molecular and Translational Medicine, University of Gothenburg, Sweden. S.T., M.K.D. and P.D.W. gratefully acknowledge the financial support of the European Regional Development Fund, Scottish Funding Council and Highlands and Islands Enterprise. Newcastle University TEM Services acknowledge BBSRC support (Grant code BB/R013942/1). The Research was supported by the National Institute for Health Research Newcastle Biomedical Research Centre based at Newcastle Hospitals NHS Foundation Trust and Newcastle University. The Newcastle Brain Tissue Resource is supported by grants from the UK Medical Research Council and the Brains for Dementia Research, a joint venture between Alzheimer’s Society and Alzheimer’s Research UK.Mutations in glucocerebrosidase (GBA) are the most prevalent genetic risk factor for Lewy body disorders (LBD)—collectively Parkinson’s disease, Parkinson’s disease dementia and dementia with Lewy bodies. Despite this genetic association, it remains unclear how GBA mutations increase susceptibility to develop LBD. We investigated relationships between LBD-specific glucocerebrosidase deficits, GBA-related pathways, and α-synuclein levels in brain tissue from LBD and controls, with and without GBA mutations. We show that LBD is characterised by altered sphingolipid metabolism with prominent elevation of ceramide species, regardless of GBA mutations. Since extracellular vesicles (EV) could be involved in LBD pathogenesis by spreading disease-linked lipids and proteins, we investigated EV derived from post-mortem cerebrospinal fluid (CSF) and brain tissue from GBA mutation carriers and non-carriers. EV purified from LBD CSF and frontal cortex were heavily loaded with ceramides and neurodegeneration-linked proteins including alpha-synuclein and tau. Our in vitro studies demonstrate that LBD EV constitute a “pathological package” capable of inducing aggregation of wild-type alpha-synuclein, mediated through a combination of alpha-synuclein–ceramide interaction and the presence of pathological forms of alpha-synuclein. Together, our findings indicate that abnormalities in ceramide metabolism are a feature of LBD, constituting a promising source of biomarkers, and that GBA mutations likely accelerate the pathological process occurring in sporadic LBD through endolysosomal deficiency.Publisher PDFPeer reviewe

An Electrochemical Detection of Metallothioneins at the Zeptomole Level in Nanolitre Volumes

An Electrochemical Detection of Metallothioneins at the Zeptomole Level in Nanolitre VolumesWe report on improvement of the adsorptive transfer stripping technique (AdTS) coupled with the differential pulse voltammetry Brdicka reaction to determine a thiol-protein. The current technique has been unable to generate reproducible results when analyzing very low sample volumes (nanolitres). This obstacle can be overcome technically by modifying the current transfer technique including cooling step of the adsorbed analyte. We tested the technique on determination of a promising tumour disease marker protein called metallothionein (MT). The detection limit (3 S/N) of MT was evaluated as 500 zeptomoles per 500 nL (1 pM) and the quantification limit (10 S/N) as 1,500 zeptomoles per 500 nL (3 pM). Further, the improved AdTS technique was utilized to analyze blood serum samples from patients with breast cancer. Based on the results obtained it can be concluded that the improved technique can be used to detect a thiolprotein in very low sample volumes and can also prevent interferences during the washing and transferring step

Elektrokemijsko određivanje metalotioneina kod domaće peradi

Metallothionein (MT) belongs to group of intracellular, low-molecular and cysteine-rich proteins with a molecular weight from 6 to 10 kDa. Owing to their high affinity to heavy metals (Zn, Cd, As, etc.) their main role is homeostatic control and detoxification of metal ions in an organism. In the present work we aimed at suggesting and utilizing electroanalytical techniques to determine content of MT in the blood serum of domestic fowls. Electrochemical measurements were performed with an AUTOLAB Analyser connected to VA-Stand 663, using a standard cell with three electrodes. Particularly, MT was detected by adsorptive transfer stripping technique in connection with differential pulse voltammetry. The detection limit of MT was estimated down to 100 fM (standards only) or down to 100 pM measured in the presence of blood serum. The average content of MT was 21.3 µM. The MT level in hens was about 25 % higher than in cocks. This phenomenon can be related to higher demands on the content of this protein in hens due the requirement for ion transport to form eggshell.Metalotionein (MT) pripada grupi intracelularnih proteina male molekularne mase bogatih cisteinom, s molekularnom masom od 6 do 10 kDa. Zbog njihovog afiniteta prema teškim metalima (Zn, Cd, As, itd.) njihova glavna uloga je homeostatska kontrola i detoksifikacija iona metala u organizmu. U ovom radu predlažu se elektroanalitičke tehnike za određivanje sadržaja MT u krvnom serumu domaće peradi. Elektrokemijska mjerenja izvršena su uređajem AUTOLAB Analyser povezanim s VA-Stand 663, koristeći standardnu ćeliju s tri elektrode. Osim toga MT je određivan tehnikom adsorptivnog transfera, povezanoj s voltmetrijom diferencijalnog pulsa. Granica detekcije MT je procjenjivana do 100 fM (samo standardi) ili do 100 pM, mjereno u prisutnosti krvnog seruma. Prosječni sadržaj MT bio je 21.3 µM. Razina MT kod kokoši bila je otprilike 25% viša nego kod pijetlova. Ta pojava može se objasniti većom potrebom za ovim proteinom kod kokoši zbog transporta iona prilikom stvaranja ljuske jajeta

A Determination of Metallothionein in Larvae of Freshwater Midges (Chironomus riparius) Using Brdicka Reaction

Among wide spectrum of biomolecules induced by various stress factors low molecular mass protein called metallothionein (MT) is suitable for assessment of the heavy metal environmental pollution. The aim of this work was to determine the metallothionein and total thiols content in larvae of freshwater midges (Chironomus riparius) sampled from laboratory exposure to cadmium(II) ions and from field studies using differential pulse voltammetry Brdicka reaction. Unique electrochemical instrument, stationary electrochemical analyser Autolab coupled with autosampler, was utilized for the analysis of the samples. The detection limit for MT was evaluated as 5 nM. The larvae exposed to two doses (50 ng/g or 50 μg/g) of cadmium(II) ions for fifteen days under laboratory controlled conditions were at the end of the exposure killed, homogenized and analysed. MT content in control samples was 1.2 μM, in larvae exposed to 50 ng Cd/g it was 2.0 μM and in larvae exposed to 50 μg Cd/g 2.9 μM. Moreover at field study chironomid larvae as well as sediment samples have been collected from eight field sites with different levels of pollution by heavy. The metals content (chromium, nickel, copper, zinc, arsenic, molybdenum, cadmium, tin and lead) in the sediment and or MT content in the chironomid larvae were determined by inductively coupled plasma mass spectrometry or Brdicka reaction, respectively