The pentameric WASH complex facilitates endosomal protein sorting by activating Arp2/3, which in turn leads to the formation of F-actin patches specifically on the endosomal surface. It is generally accepted that WASH complex attaches to the endosomal membrane via the interaction of its subunit FAM21 with the retromer subunit VPS35. However, we observe the WASH complex and F-actin present on endosomes even in the absence of VPS35. We show that the WASH complex binds to the endosomal surface in both a retromer-dependent and a retromer-independent manner. The retromer-independent membrane anchor is directly mediated by the subunit SWIP. Furthermore, SWIP can interact with a number of phosphoinositide species. Of those, our data suggest that the interaction with phosphatidylinositol-3,5-bisphosphate (PI(3,5)P2 ) is crucial to the endosomal binding of SWIP. Overall, this study reveals a new role of the WASH complex subunit SWIP and highlights the WASH complex as an independent, self-sufficient trafficking regulator.
This review summarizes the current knowledge on essential vitamins B1, B2, B3, and B5. These B-complex vitamins must be taken from diet, with the exception of vitamin B3, that can also be synthetized from amino acid tryptophan. All of these vitamins are water soluble, which determines their main properties, namely: they are partly lost when food is washed or boiled since they migrate to the water; the requirement of membrane transporters for their permeation into the cells; and their safety since any excess is rapidly eliminated via the kidney. The therapeutic use of B-complex vitamins is mostly limited to hypovitaminoses or similar conditions, but, as they are generally very safe, they have also been examined in other pathological conditions. Nicotinic acid, a form of vitamin B3, is the only exception because it is a known hypolipidemic agent in gram doses. The article also sums up: (i) the current methods for detection of the vitamins of the B-complex in biological fluids; (ii) the food and other sources of these vitamins including the effect of common processing and storage methods on their content; and (iii) their physiological function.
BACKGROUND: Tuberous sclerosis complex (TSC), a multi-system genetic disorder often associated with autism spectrum disorder (ASD), is caused by mutations of TSC1 or TSC2, which lead to constitutive overactivation of mammalian target of rapamycin (mTOR). In several Tsc1+/- and Tsc2+/- animal models, cognitive and social behavior deficits were reversed by mTOR inhibitors. However, phase II studies have not shown amelioration of ASD and cognitive deficits in individuals with TSC during mTOR inhibitor therapy. We asked here if developmental epilepsy, common in the majority of individuals with TSC but absent in most animal models, could explain the discrepancy. METHODS: At postnatal day P12, developmental status epilepticus (DSE) was induced in male Tsc2+/- (Eker) and wild-type rats, establishing four experimental groups including controls. In adult animals (n = 36), the behavior was assessed in the paradigms of social interaction test, elevated plus-maze, light-dark test, Y-maze, and novel object recognition. The testing was carried out before medication (T1), during a 2-week treatment with the mTOR inhibitor everolimus (T2) and after an 8-week washing-out (T3). Electroencephalographic (EEG) activity was recorded in a separate set of animals (n = 18). RESULTS: Both Tsc2+/- mutation and DSE caused social behavior deficits and epileptiform EEG abnormalities (T1). Everolimus led to a persistent improvement of the social deficit induced by Tsc2+/-, while deficits related to DSE did not respond to everolimus (T2, T3). CONCLUSIONS: These findings may contribute to an explanation why ASD symptoms in individuals with TSC, where comorbid early-onset epilepsy is common, were not reliably ameliorated by mTOR inhibitors in clinical studies.
- MeSH
- Autistic Disorder * MeSH
- Haploinsufficiency MeSH
- Rats MeSH
- Status Epilepticus * MeSH
- TOR Serine-Threonine Kinases genetics MeSH
- Tuberous Sclerosis Complex 2 Protein genetics MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Male MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
The mechanism of cyanide's inhibitory effect on the mitochondrial cytochrome c oxidase (COX) as well as the conditions for its recovery have not yet been fully explained. We investigated three parameters of COX function, namely electron transport (oxygen consumption), proton transport (mitochondrial membrane potential Δψ(m)) and the enzyme affinity to oxygen (p₅₀ value) with regard to the inhibition by KCN and its reversal by pyruvate. 250 μM KCN completely inhibited both the electron and proton transport function of COX. The inhibition was reversible as demonstrated by washing of mitochondria. The addition of 60 mM pyruvate induced the maximal recovery of both parameters to 60-80% of the original values. When using low KCN concentrations of up to 5 μM, we observed a profound, 30-fold decrease of COX affinity for oxygen. Again, this decrease was completely reversed by washing mitochondria while pyruvate induced only a partial, yet significant recovery of oxygen affinity. Our results demonstrate that the inhibition of COX by cyanide is reversible and that the potential of pyruvate as a cyanide poisoning antidote is limited. Importantly, we also showed that the COX affinity for oxygen is the most sensitive indicator of cyanide toxic effects.
- MeSH
- Liver metabolism MeSH
- Rats MeSH
- Potassium Cyanide pharmacology MeSH
- Pyruvic Acid metabolism MeSH
- Oxygen metabolism MeSH
- Membrane Potential, Mitochondrial physiology MeSH
- Mitochondria metabolism MeSH
- Rats, Wistar MeSH
- Protons MeSH
- Electron Transport Complex IV antagonists & inhibitors metabolism MeSH
- Oxygen Consumption physiology MeSH
- Electron Transport physiology MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Male MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Exploring the structure and function of protein complexes requires their isolation in the native state-a task that is made challenging when studying labile and/or low abundant complexes. The difficulties in preparing membrane-protein complexes are especially notorious. The cyanobacterium Synechocystis sp. PCC 6803 is a widely used model organism for the physiology of oxygenic phototrophs, and the biogenesis of membrane-bound photosynthetic complexes has traditionally been studied using this cyanobacterium. In a typical approach, the protein complexes are purified with a combination of His-affinity chromatography and a size-based fractionation method such as gradient ultracentrifugation and/or native electrophoresis. However, His-affinity purification harbors prominent contaminants and the levels of many proteins are too low for a feasible multi-step purification. Here, we have developed a purification method for the isolation of 3x FLAG-tagged proteins from the membrane and soluble fractions of Synechocystis. Soluble proteins or solubilized thylakoids are subjected to a single affinity purification step that utilizes the highly specific binding of FLAG-affinity resin. After an intensive wash, the captured proteins are released from the resin under native conditions using an excess of synthetic 3x FLAG peptide. The protocol allows fast isolation of low abundant protein complexes with a superb purity.
- Publication type
- Journal Article MeSH
A series of 2-piperazin-1-yl-quinazolines were synthesized and evaluated for their antiaggregative activity. The synthesized small molecule compounds have potently inhibited platelet aggregation in vitro and blocked FITC-Fg binding to αIIbβ3 integrin in a suspension of washed human platelets. The key αIIbβ3 protein-ligand interactions were determined in docking experiments and some correlations have been observed between values of the affinity and docking scores.
- MeSH
- Platelet Aggregation drug effects MeSH
- Quinazolines chemistry pharmacology MeSH
- Platelet Aggregation Inhibitors chemistry pharmacology MeSH
- Humans MeSH
- Ligands MeSH
- Piperazines chemistry pharmacology MeSH
- Molecular Docking Simulation MeSH
- Platelet Glycoprotein GPIIb-IIIa Complex metabolism MeSH
- Blood Platelets cytology drug effects metabolism MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Native polyacrylamide electrophoresis in the presence of two reversible protein anionic stains (Ponceau S and Ponceau 2R) was used to study the oligomeric states of soluble proteins. A mild binding of the used protein stains to nondissociated protein oligomers imposed a charge shift on the proteins resulting into separation of protein species according to their size under physiological conditions. Adsorbed stains could be easily removed after electrophoresis by washing of polyacrylamide gel with buffer and protein complexes could be visualized either by the detection of their enzyme activity or by using a nonspecific protein stain. The specific detection of enzyme activity of glycosidases, lactate dehydrogenase, or phosphatases was shown as an example.
Předmět sdělení: Druhý oddíl literárního přehledu o důkazech podporujících současnou strategii prevence zubního kazu shrnuje poznatky o různých aplikačních formách fluoridů a jejich účinku, zejména u dětí a mládeže. Oddíl je rozčleněn na formy systémové fluoridace, zahrnující fluoridaci pitné vody a mléka, fluoridové suplementy a lokální aplikační formy, zubní pasty, ústní vody, fluoridové gely a laky, prostředky pomalu uvolňující fluorid a komplexní fluoridové soli stříbra. Citované údaje o jejich účinku jsou převzaty zejména z metaanalytických studií a zpráv z cochranovské databáze systematických review. Zmiňovány jsou dále základní dokumenty Světové zdravotnické organizace a národních i nadnárodních odborných organizací, které upozorňují na nutnost kvalifikovaného řízení fluoridové prevence na individuální i komunitní úrovni k dosažení maximálního účinku a minimalizace rizik. Účinnost (ale i bezpečnost) lokálně aplikovaných prostředků v individuální domácí péči o chrup je závislá na míře compliance jedinců, respektive rodičů dětí a na míře kompetence poskytovatelů preventivního poradenství. Široké spektrum těchto prostředků umožňuje individualizaci fluoridové prevence na základě analýzy rizika kazivé ataky a s přihlédnutím k dalším preventivním opatřením.
Background: The second part of the literature review presents evidences supporting the current strategy of caries prevention summarizes knowledge about different forms of fluoride and their effects, especially in children and youth. The review describes individual forms of systemic fluoridation comprising fluoridation of drinking water, milk and fluoride supplements, and topical forms comprising toothpastes, mouthwashes, fluoride gels and varnishes, slow release fluoride devices and complex fluoride salts of silver. The cited data on their effect are taken mainly from meta-analytic studies and reports from Cochrane database systematic reviews. Mentioned below are the basic documents of the World Health Organization and national and international professional organizations, which point to the need for the qualified management of fluoride prevention at the individual and community level to achieve maximum effect and risk minimization. Efficiency (but safety) of topically applied fluorides in individual home dental care is dependent on the degree of compliance of individuals, respectively parents of children and on the level of competence of providers of preventive counselling. The broad spectrum of these resources allows individualization of fluoride prevention based on risk analysis of caries attack and taking into account other preventive measures.
- Keywords
- systémová fluoridová prevence, lokální fluoridová prevence,
- MeSH
- Self Administration MeSH
- Fluorosis, Dental etiology MeSH
- Fluoridation * MeSH
- Fluorides * administration & dosage adverse effects therapeutic use MeSH
- Gels MeSH
- Cariostatic Agents administration & dosage adverse effects therapeutic use MeSH
- Sodium Chloride, Dietary MeSH
- Delayed-Action Preparations MeSH
- Humans MeSH
- Fluorides, Topical administration & dosage adverse effects therapeutic use MeSH
- Evidence-Based Medicine MeSH
- Milk MeSH
- Oral Hygiene MeSH
- Drinking Water MeSH
- Dietary Supplements MeSH
- Primary Prevention MeSH
- Tablets administration & dosage MeSH
- Mouthwashes MeSH
- Dose-Response Relationship, Drug MeSH
- Dental Caries * prevention & control MeSH
- Dental Materials chemistry MeSH
- Toothpastes administration & dosage chemistry MeSH
- Check Tag
- Humans MeSH
- Publication type
- Research Support, Non-U.S. Gov't MeSH
- Review MeSH
BACKGROUND: p-aminobenzamidine (p-ABA) is used as a ligand in the purification of many serine proteases and in their removal from heterogeneous samples. Moreover, p-ABA has a potent ability to bind Ca(2+)-binding proteins. The binding ability and use of p-ABA in purification processes is still not fully understood. METHODOLOGY/PRINCIPAL FINDINGS: A p-Aminobenzamidine (p-ABA) ligand enabled the purification of the panallergenic proteins tropomyosin and paramyosin, as well as actin, tubulin, troponin and several kinases and annexins, with variable specificity depending on the tissue source and slight modifications to the purification process. The high affinity of p-ABA to tropomyosin, paramyosin, actin, troponin and myosin is calcium-dependent, since calcium regulates the function of these proteins. In addition, p-ABA probably simulates phosphorylated serine and therefore purified appropriate kinases. Because p-ABA binds to calcium-dependent proteins, and probably those with binding sites containing serine, it is not a suitable inhibitor of proteolysis during the purification of such proteins. p-ABA is widely used to inhibit proteases during protein purification processes, but it is used in columns here to purify non-protease proteins. Two strategies were applied; the first was the inactivation of proteases that were not of interest using protease inhibitors. The second strategy employed was the use of a Ca(2+) wash solution to remove calcium-dependent proteins. The removal of calcium-dependent proteins from rabbit hind muscle pointed out even more selective purification. It is possible to obtain two purified samples: a) calcium dependent proteins and b) calcium independent proteins. Moreover, p-ABA may be useful as a model to study processes involving the phosphorylation of serine. CONCLUSION: A p-Aminobenzamidine (p-ABA) ligand enabled the purification of non-protease proteins, with variable specificity depending on the tissue source and slight modifications to the purification process. The method is applicable to various scientific branches, but is especially practical for medicinal applications.
- MeSH
- Electrophoresis, Gel, Two-Dimensional MeSH
- Actins isolation & purification MeSH
- Phosphotransferases isolation & purification MeSH
- Spectrometry, Mass, Electrospray Ionization MeSH
- Rabbits MeSH
- Proteins isolation & purification MeSH
- Proteomics methods MeSH
- Mites MeSH
- Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization MeSH
- Muscles metabolism MeSH
- Tropomyosin isolation & purification MeSH
- Troponin isolation & purification MeSH
- Tubulin isolation & purification MeSH
- Calcium metabolism MeSH
- Animals MeSH
- Check Tag
- Rabbits MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Nozokomiální nákazy (NN) vedou k prodloužení doby hospitalizace, zvýšení nákladů na terapii, zvýšení nákladů na pobyt v nemocnici a snížení práceschopnosti. Představují tak závažný problém v oblasti veřejného zdraví. Hygienicko-epidemiologická péče se zaměřuje zejména na infekce s častým výskytem, případně závažnými důsledky pro pacienta. Základním předpokladem pro nastavení účinných preventivních postupů je adekvátní surveillance, tedy komplexní epidemiologický přístup spočívající v aktivním získávání a následném zpracováním informací o povaze a charakteru nákaz, procesu jejich šíření, včetně stanovení opatření na konkrétním pracovišti. Prevenci NN v praxi praktického lékaře (PL) je třeba věnovat stejnou pozornost jako v jakýchkoliv jiných zdravotnických zařízeních, navíc s přihlédnutím k charakteru poskytované péče. Zejména krátká doba kontaktu s pacientem může vést k podcenění rizika vzniku NN, která se v praxi projeví většinou po opuštění ordinace PL ať již v domácí péči, či během eventuální následné hospitalizace. Základní informace o povaze a charakteru nejčastějších či pro pacienta nejzávažnějších NN, včetně povinností vyplývajících z legislativy, by měly být dostupné všem zdravotnických pracovníkům.
Nosocomial infections (NI) lead to an extension of hospital stay, an increase in treatment costs and an increase in general hospitalisation expenses, as well as increasing the time the patient is out of work. NI represent a severe public health problem. Hygienic-epidemiological treatment is focused mainly on the frequent infections, or infections with the serious consequences for the patients. The main step towards implementing the efficient precautions is the adequate surveillance, as the complex epidemiological approach consisting of active monitoring, analysis of the relevant information concerning infection characteristics and spread, including feedback leading to the practical measurements in the given workplace. NI precautions in the general practitioner’s office should implement its specifics. Mainly the short contact with the patient could lead to the undervaluation of the NI risk, as most of the NI is developed consequently, during the after-care or the hospitalisation. Every health professional should know basic information concerning NI, precaution and related legislation.
