Morphological characteristics of cultured fresh and thawed pericardium cells

The need for selection of the optimal material for the manufacturing of cardio-patches can be resolved by the use of cryostored autologous pericardial tissue. This short communication is a concise fragment of a large-scale research and demonstrates only the efficiency of cell culturing before and after pericardial preservation in the low temperature conditions.     

Nonrandom distribution of goblet cells around the circumference of colonie crypt

The ascending colon of the guinea pig was cut serially and stained with the periodic-acid-Schiff-hematoxylin method. Maps of transversely sectioned crypts were prepared using a microscopic eye-piece projector. The number and angular positions of goblet cells were mapped around the circumference of transverse sections of the crypt. A method of the "statistics of the circumference" was introduced in order to assess the pattern of distribution of goblet cells. This distribution was found to be significantly nonrandom. The nonrandomness is such that the goblet cells tend to occupy positions of the crypt circumference in maximal distances from each other.     

Quantitative Proteomic Analysis in Metastatic Renal Cell Carcinoma Reveals a Unique Set of Proteins with Potential Prognostic Significance

Metastatic renal cell carcinoma (RCC) is one of the most treatment-resistant malignancies, and patients have a dismal prognosis, with a <10% five-year survival rate. The identification of markers that can predict the potential for metastases will have a great effect in improving patient outcomes. In this study, we used differential proteomics with isobaric tags for relative and absolute quantitation (iTRAQ) labeling and LC-MS/MS analysis to identify proteins that are differentially expressed in metastatic and primary RCC. We identified 1256 non-redundant proteins, and 456 of these were quantified. Further analysis identified 29 proteins that were differentially expressed (12 overexpressed and 17 underexpressed) in metastatic and primary RCC. Dysregulated protein expressions of profilin-1 (Pfn1), 14–3-3 zeta/delta (14–3-3ζ), and galectin-1 (Gal-1) were verified on two independent sets of tissues by means of Western blot and immunohistochemical analysis. Hierarchical clustering analysis showed that the protein expression profile specific for metastatic RCC can distinguish between aggressive and non-aggressive RCC. Pathway analysis showed that dysregulated proteins are involved in cellular processes related to tumor progression and metastasis. Furthermore, preliminary analysis using a small set of tumors showed that increased expression of Pfn1 is associated with poor outcome and is a potential prognostic marker in RCC. In addition, 14–3-3ζ and Gal-1 also showed higher expression in tumors with poor prognosis than in those with good prognosis. Dysregulated proteins in metastatic RCC represent potential prognostic markers for kidney cancer patients, and a greater understanding of their involved biological pathways can serve as the foundation of the development of novel targeted therapies for metastatic RCC.Renal cell carcinoma (RCC)¹ is the most common neoplasm of the adult kidney. Worldwide incidence and mortality rates of RCC are rising each decade (1). Seventy-five percent of kidney tumors are of the clear cell (ccRCC) subtype (2). Although modern imaging techniques for abdominal screening have led to increased incidental detection of renal tumors (3), unfortunately ∼25% to 30% of patients still have metastases at presentation.The prognosis with RCC is quite variable. The greatest risk of recurrence following nephrectomy is within the first 3 to 5 years (4). The ability to predict which tumors will metastasize would have a significant effect on patient outcomes, because the likelihood of a favorable response to treatment is greater when the metastatic burden is limited, and surgical resection of a single or limited number of metastases can result in longer survival (5). Furthermore, ∼3% of patients will develop a second primary renal tumor, either synchronous or metachronous. Currently, patient prognosis is assessed based on histological parameters and a multivariate analysis developed at Memorial Sloan Kettering (6), but neither is sufficiently accurate. A more accurate assessment of prognosis is urgently needed to better guide patient management.Although surgery can be curative for localized disease, many patients eventually relapse. Metastatic RCC is one of the most treatment-resistant malignancies, with chemotherapy and radiotherapy having limited effect. The five-year survival rate for metastatic RCC is ≤10% (7). Although there has been much progress in RCC treatment with the new era of antiangiogenic therapy, the majority of patients ultimately suffer a relapse and die from progression of the cancer. A more in-depth understanding of the pathogenesis of metastasis will be a cornerstone in the development of new targeted therapies. A number of prognostic markers have previously been identified based on comparative analysis of primary and metastatic tumors, including C-reactive protein, tetraspanin 7, hypoxia-inducible factor 1 α, phos-S6, U3 small nucleolar ribonucleoprotein protein, carbonic anhydrase IX, and microvascular density (8–14). However, no biomarker has yet had an established clinical role independent of stage (15). Differential protein expression between primary RCC and normal tissues was previously studied (16–18). Also, differential expression between primary and metastatic kidney disease has been investigated at the microRNA level (19, 20). Molecular analyses hold the promise of providing a better understanding of the pathogenesis of kidney cancer (21).In this study, we aimed to elucidate the pathogenesis of RCC metastasis through proteomic analysis and to identify potential prognostic markers for kidney cancer. We performed quantitative proteomic analysis using isobaric tags for relative and absolute quantitation (iTRAQ) labeling and LC-MS/MS to identify proteins that were dysregulated in metastatic RCC relative to primary RCC. Differential expressions of selected biologically interesting proteins—profilin-1 (Pfn1), 14–3-3 zeta/delta (14–3-3ζ), and galectin-1 (Gal-1)—were validated on two independent sets of tumors by means of western blot (WB) analysis and immunohistochemistry (IHC). Hierarchical clustering analysis showed that differential protein expression can distinguish between aggressive and non-aggressive tumors. In order to explore the role of these dysregulated proteins in tumor progression, we performed Gene Ontology (GO) and pathway analyses. In addition, we carried out a preliminary analysis to assess the potential of Pfn1, 14–3-3ζ, and Gal-1 as prognostic markers in RCC.     

Trade in the Decade Following the Collapse of the USSR

In Soviet times, Moscow was a consumers’ oasis in a country of rampant scarcity. Millions of workers and peasants from all the free republics met in the capital of the unbreakable Union to stock up on delicacies, foreign goods, and all the things not available in the national hinterland. As new markets with a certain degree of specialization emerged, this introduced more stringent terms of trade and other measures aimed at eliminating the negative impact of commerce on the environment and improving the sanitary condition of the area, as well as improving traffic conditions and the general appearance of the environment. The authors analyze the space of commerce during the Soviet Union’s existence and after its collapse, and show how popular attitudes changed.     

Universal soldier: Pseudomonas aeruginosa — an opportunistic generalist

The opportunistic pathogen Pseudomonas aeruginosa commonly causes chronic and ultimately deadly lung infections in individuals with the genetic disease cystic fibrosis (CF). P. aeruginosa is metabolically diverse; it displays a remarkable ability to adapt to and successfully occupy almost any niche, including the ecologically complex CF lung. These P. aeruginosa lung infections are a fascinating example of microbial evolution within a “natural” ecosystem. Initially, P. aeruginosa shares the lung niche with a plethora of other microorganisms and is vulnerable to antibiotic challenges. Over time, adaptive evolution leads to certain commonly-observed phenotypic changes within the P. aeruginosa population, some of which render it resistant to antibiotics and apparently help it to out-compete the other species that co-habit the airways. Improving genomics techniques continue to elucidate the evolutionary mechanisms of P. aeruginosa within the CF lung and will hopefully identify new vulnerabilities in this robust and versatile pathogen.     

Chromosomal aberration frequencies determined by conventional methods: Parallel increases over time in the region of a petrochemical industry and throughout the Czech Republic

The rationale for cytogenetic monitoring to determine if safe maximum allowable concentrations (MAC) of genotoxic chemicals are being maintained in a workplace is that exposure levels that do not increase chromosomal aberration frequencies are without harmful effects. Such monitoring, widely used in occupational health programs in the Czech Republic (CR), includes workers exposed to 1,3-butadiene (BD) or other chemicals. Studies of BD exposed workers in the years 1992, 1993, 1994, 1998, and 2004 compared mean frequencies of cells carrying chromosomal aberrations (frequency of aberrant cells=%AB.C.) in exposed workers with those in non-exposed matched controls in the same plant or in other individuals living in the region of the same petrochemical industry. Workers potentially exposed to acrylonitrile at this site were also evaluated in 2000, along with another unexposed matched control group. The %AB.C. values of exposed workers and their controls were also compared with reference values determined for normal individuals (ages 20-59 years) throughout the CR. Substantial discrepancies were noted between subjects in the region of the petrochemical industry (exposed workers and controls) for the years 2000 and 2004 and the reference CR-wide normal values that had been determined during an earlier time period. The matched non-exposed controls at the petrochemical industry site showed a mean %AB.C. value of 1.56+/-1.23% (N=25) in 1998; this rose to a mean of 2.65+/-2.29% (N=33) in 2000. In 2004, values for non-exposed matched controls at the industry site were 2.64+/-1.75% for males (N=25) and 2.38+/-1.74% (N=26) for females. However, the earlier determined CR-wide %AB.C. mean reference values for normal individuals were 1.77+/-1.16% (N=1305) for the interval 1977-1988 and 1.45+/-1.17% (N=2140) for the interval 1991-1999. As both reference values are substantially lower than those determined in 2000 and 2004 for the non-exposed matched controls at the petrochemical industry site, an analysis of the CR-wide mean normal individual reference values for this same 2000-2004 period was conducted. Unexpectedly, it was found that this reference value too had risen to 1.95+/-1.36% (N=1045) and was comparable to the concurrent matched control values at the petrochemical industry site where the monitoring studies were conducted. This substantial increase in %AB.C. values in 2000 and 2004, therefore, has occurred throughout the CR and is probably unrelated to chemicals uniquely present at the petrochemical industry site.     

Imprinted polymer layer for recognizing double-stranded DNA

A method of preparing a thin polymer layer able to recognize double-stranded DNA (dsDNA) was developed by using 2-vinyl-4,6-diamino-1,3,5-triazine (VDAT) as a functional monomer for creating a DNA-imprinted polymer. The formation of hydrogen bonds between VDAT and A-T base pairs in dsDNA was confirmed by measuring the effects of VDAT on the melting point and the NMR and CD spectra of dsDNA. An imprinted polymer that can recognize dsDNA of the verotoxin gene was prepared by polymerizing VDAT, acrylamide, a crosslinking agent, and the template verotoxin dsDNA on a silanized glass surface. The specificity of this polymer layer for binding verotoxin dsDNA was investigated by using fluorescent-labelled dsDNAs. The fluorescence intensity of the polymer layer after binding verotoxin dsDNA was twice as high as after binding oligo(dG)-oligo(dC), indicating that verotoxin dsDNA was preferentially bound to the polymer imprinted with verotoxin dsDNA. The kinetics of verotoxin dsDNA binding to the imprinted polymer were analyzed by surface plasmon resonance measurements. The dissociation constant (KD) was low, of the order of 10(-9)M.     

Induction by beta-bungarotoxin of apoptosis in cultured hippocampal neurons is mediated by Ca(2+)-dependent formation of reactive oxygen species

The component of the venom of the Taiwanese banded krait Bungarus multicinctus, beta-bungarotoxin (beta-BuTx), acts as an extremely potent inducer of neuronal apoptosis when applied to rat hippocampal cultures. While induction of cell death is dependent on toxin binding to voltage-activated K+ channels and subsequent internalization, the pro-apoptotic signals triggered by picomolar concentrations of beta-BuTx are not understood. Following toxin binding, a dramatic increase in intracellular Ca2+ became detectable after 30 min, and in reactive oxygen species (ROS) after 3-4 h. Conversely, Ca2+ chelators, radical quenchers and antioxidants efficiently antagonized beta-BuTx induced apoptosis. As shown for the antioxidant 2,3-dihydroxybenzoic acid, analysis by matrix assisted laser desorbtion-time of flight (MALDI-TOF) mass spectrometry excluded the protective effects to be due to reductive cleavage of the toxic beta-BuTx dimer. Inhibitors of the intracellular antioxidant defence system enhanced neuronal susceptibility to beta-BuTx, supporting the essential role of ROS in beta-BuTx-initiated apoptosis. Cell damage was accompanied by an accumulation of markers of oxidative cell stress, phospholipid hydroxyperoxides and the lipid peroxidation product, malonyl dialdehyde. These observations indicate that beta-BuTx-induced cell death resulted from an intracellular signalling cascade involving subsequent stages of a dramatic rise in free Ca2+, the accumulation of ROS, membrane lipid peroxidation and, finally, apoptosis.     

Subcellular localization and regulation of coenzyme A synthase

CoA synthase mediates the last two steps in the sequence of enzymatic reactions, leading to CoA biosynthesis. We have recently identified cDNA for CoA synthase and demonstrated that it encodes a bifunctional enzyme possessing 4'-phosphopantetheine adenylyltransferase and dephospho-CoA kinase activities. Molecular cloning of CoA synthase provided us with necessary tools to study subcellular localization and the regulation of this bifunctional enzyme. Transient expression studies and confocal microscopy allowed us to demonstrate that full-length CoA synthase is associated with the mitochondria, whereas the removal of the N-terminal region relocates the enzyme to the cytosol. In addition, we showed that the N-terminal sequence of CoA synthase (amino acids 1-29) exhibits a hydrophobic profile and targets green fluorescent protein exclusively to mitochondria. Further analysis, involving subcellular fractionation and limited proteolysis, indicated that CoA synthase is localized on the mitochondrial outer membrane. Moreover, we demonstrate for the first time that phosphatidylcholine and phosphatidylethanolamine, which are the main components of the mitochondrial outer membrane, are potent activators of both enzymatic activities of CoA synthase in vitro. Taken together, these data provide the evidence that the final stages of CoA biosynthesis take place on mitochondria and the activity of CoA synthase is regulated by phospholipids.