A Screen for Endocytic Motifs

Sorting signals for cargo selection into coated vesicles are usually in the form of short linear motifs. Three motifs for clathrin‐mediated endocytosis have been identified: YXXΦ, [D/E]XXXL[L/I] and FXNPXY. To search for new endocytic motifs, we made a library of CD8 chimeras with random sequences in their cytoplasmic tails, and used a novel fluorescence‐activated cell sorting (FACS)‐based assay to select for endocytosed constructs. Out of the five tails that were most efficiently internalized, only one was found to contain a conventional motif. Two contain dileucine‐like sequences that appear to be variations on the [D/E]XXXL[L/I] motif. Another contains a novel internalization signal, YXXXΦN, which is able to function in cells expressing a mutant µ2 that cannot bind YXXΦ, indicating that it is not a variation on the YXXΦ motif. Similar sequences are present in endogenous proteins, including a functional YXXXΦN (in addition to a classical YXXΦ) in cytotoxic T‐lymphocyte‐associated protein 4 (CTLA‐4). Thus, the repertoire of endocytic motifs is more extensive than the three well‐characterized sorting signals.     

Multivariate proteomic profiling identifies novel accessory proteins of coated vesicles

Despite recent advances in mass spectrometry, proteomic characterization of transport vesicles remains challenging. Here, we describe a multivariate proteomics approach to analyzing clathrin-coated vesicles (CCVs) from HeLa cells. siRNA knockdown of coat components and different fractionation protocols were used to obtain modified coated vesicle-enriched fractions, which were compared by stable isotope labeling of amino acids in cell culture (SILAC)-based quantitative mass spectrometry. 10 datasets were combined through principal component analysis into a "profiling" cluster analysis. Overall, 136 CCV-associated proteins were predicted, including 36 new proteins. The method identified >93% of established CCV coat proteins and assigned >91% correctly to intracellular or endocytic CCVs. Furthermore, the profiling analysis extends to less well characterized types of coated vesicles, and we identify and characterize the first AP-4 accessory protein, which we have named tepsin. Finally, our data explain how sequestration of TACC3 in cytosolic clathrin cages causes the severe mitotic defects observed in auxilin-depleted cells. The profiling approach can be adapted to address related cell and systems biological questions.     

Season dependent response of the Marsh frog (Rana ridibunda) to cadmium exposure

Cadmium toxicity related to cysteine metabolism and glutathione levels in several tissues of the Marsh frog (Rana ridibunda) collected in late spring were investigated after exposure to 80 mg CdCl2 L(-1) for 168 h. The results were compared to those obtained in a previous experiment carried out in autumn. The most striking changes involved the brain which could not maintain a proper glutathione level and the testes in which neither GSH nor sulfane sulfur levels recovered. Substantial damage is expected in the presence of Cd which decreases the antioxidant status of these tissues. It seems that in spring, frogs had lesser tolerance for Cd in comparison with frogs in autumn. This may be caused by the transition to aerobic respiration after hibernation.     

A novel mutation in FGFR3 causes camptodactyly, tall stature, and hearing loss (CATSHL) syndrome

Activating mutations of FGFR3, a negative regulator of bone growth, are well known to cause a variety of short-limbed bone dysplasias and craniosynostosis syndromes. We mapped the locus causing a novel disorder characterized by camptodactyly, tall stature, scoliosis, and hearing loss (CATSHL syndrome) to chromosome 4p. Because this syndrome recapitulated the phenotype of the Fgfr3 knockout mouse, we screened FGFR3 and subsequently identified a heterozygous missense mutation that is predicted to cause a p.R621H substitution in the tyrosine kinase domain and partial loss of FGFR3 function. These findings indicate that abnormal FGFR3 signaling can cause human anomalies by promoting as well as inhibiting endochondral bone growth.     

Verteporfin, photofrin II, and merocyanine 540 as PDT photosensitizers against melanoma cells

The efficiency of photodynamic effect (PDE) for Photofrin II (PfII), Verteporfin, and Merocyanine 540 (MC540) was compared against neoplastic cells. Triplet state lifetimes and singlet molecular oxygen quantum yields were correlated with biological effect. PfII triplet lifetime was two times longer than that of Verteporfin, however, its singlet molecular oxygen quantum yield was two times lower in comparison with Verteporfin. High singlet molecular oxygen quantum yield of Verteporfin resulted in high biological efficacy. To achieve 50% mortality of cells four times lower light dose and five times lower concentration of Verteporfin were applied in comparison with PfII. The same level of cell damage was reached using 10 times higher light dose and two times higher concentration of MC540 in comparison with PfII. Our results confirm that singlet molecular oxygen based mechanism, prevalent for Verteporfin and PfII, was highly effective against melanoma cells. Verteporfin can be used at small doses with high cellular damage efficiency.     

A Preliminary Study Showing the Impact of Genetic and Dietary Factors on GC–MS-Based Plasma Metabolome of Patients with and without PROX1-Genetic Predisposition to T2DM up to 5 Years Prior to Prediabetes Appearance

Risk factors for type 2 diabetes mellitus (T2DM) consist of a combination of an unhealthy, imbalanced diet and genetic factors that may interact with each other. Single nucleotide polymorphism (SNP) in the prospero homeobox 1 (PROX1) gene is a strong genetic susceptibility factor for this metabolic disorder and impaired β-cell function. As the role of this gene in T2DM development remains unclear, novel approaches are needed to advance the understanding of the mechanisms of T2DM development. Therefore, in this study, for the first time, postprandial changes in plasma metabolites were analysed by GC–MS in nondiabetic men with different PROX1 genotypes up to 5 years prior to prediabetes appearance. Eighteen contestants (12 with high risk (HR) and 6 with low risk (LR) genotype) participated in high-carbohydrate (HC) and normo-carbohydrate (NC) meal-challenge tests. Our study concluded that both meal-challenge tests provoked changes in 15 plasma metabolites (amino acids, carbohydrates, fatty acids and others) in HR, but not LR genotype carriers. Postprandial changes in the levels of some of the detected metabolites may be a source of potential specific early disturbances possibly associated with the future development of T2DM. Thus, accurate determination of these metabolites can be important for the early diagnosis of this metabolic disease.     

Towards new boron carriers for boron neutron capture therapy: metallacarboranes and their nucleoside conjugates

Thymidine conjugates containing metallacarborane, {8-[5-(N(3)-thymidine)-3-oxa-pentoxy]-3-cobalt bis(1,2-dicarbollide)}- (5) and {8-[5-(O(4)-thymidine)-3-oxa-pentoxy]-3-cobalt bis(1,2-dicarbollide)}- (6) ions and several simple [3-cobalt bis(1,2-dicarbollide)]- ion (1) derivatives have been studied as potential boron carriers for BNCT. Compound 6 and some nonnucleoside derivatives of 1 were not toxic above 100 microM. The partition coefficient for both metallacarborane bearing thymidine conjugates 5 and 6 was more than 500 times higher than that of unmodified nucleoside. The cellular uptake studies showed accumulation of compounds 6 in V79 Chinese hamster cells but not of compound 5. The low toxicity of conjugate type of 6 together with its high partition coefficient suggest that judicially designed derivatives of metallacarboranes can be considered as potential boron carriers for BNCT.     

Vascular endothelial growth factor--structure and functions

Vascular endothelial cell growth factor (VEGF), originally described as a vascular permeability factor, is currently known as one of the main factors which regulate angiogenesis. It plays an important role in the regulation of normal as well as pathological angiogenesis. In this paper we try to shortly review the actual knowledge on VEGF protein family, its expression, VEGF receptors and role of VEGF in signal transduction. The aim of this review is also to summarize recent achievements in research on biological functions of vascular endothelial growth factor and their clinical applications.     

Immunohistochemical evaluation of cell proliferation and apoptosis markers in ovarian surface epithelial cells of cladribine-treated rats

Cladribine has been used in the treatment of hairy cell leukemia for about 30 years. In addition, the number of indications for the application of 2-CdA is constantly increasing. The treatment with cladribine, of younger persons and even children, appears to be a major factor stimulating the more exact recognition of its activities. However, till now, little has been known about the impact of cladribine on the reproductive system. The aim of the study was to evaluate the immunohistochemical expression of cell proliferation and apoptosis markers in ovarian surface epithelial (OSE) cells. In our study, ten rats were placed into two equal groups. The study group received daily subcutaneous injections of cladribine in a dose of 0.10 mg/kg of weight/day for one cycle lasting 7 days. The control group received only saline injections. The rats were sacrificed 24 h after the last injection, and their ovaries were extracted. The sections were immunohistochemically stained with cell proliferation marker Ki-67 and the apoptosis marker caspase 3. The expressions of the markers were evaluated using a light microscope. An analysis was made using an image analysis system and the CellAD software. The results were then statistically explored by way of the Mann–Whitney U test. The proliferative index (Ki-67) of ovarian surface epithelial cells was significantly lower in the study group than in the control group (p?<?0.05). These results suggest that cladribine treatment has a potential to inhibit the OSE cell proliferation in rats. The apoptosis marker demonstrated a significant increase after the cladribine treatment. These suggest that cladribine induces apoptosis in OSE cells.