Monitoring liver alterations during hepatic tumorigenesis by NMR profiling and pattern recognition

Human hepatocellular carcinoma (HCC) is the most recurrent malignancy of the liver and represents one of the main causes of cancer death worldwide. Furthermore, the liver is the most frequent site of metastatic colonization, and hepatic metastases are far more common than primary cancers in Western countries. A possible way of investigating liver diseases is to study the tissue metabolic profiles. High-resolution nuclear magnetic resonance (NMR) spectroscopy of hepatic tissue extracts was combined with pattern-recognition and visualization techniques to uncover metabolic differences among analyzed tissue types. Extracts were from primary HCC, chronic hepatitis-C-virus related cirrhotic tissues, hepatic metastases from colorectal carcinomas, and non-cirrhotic normal liver tissues adjacent to metastases as controls. We identified all metabolites present in the NMR spectra, and after statistical evaluation of all spectral classes, we were able to define the metabolic changes underlying the different liver conditions and diseases. In particular, the lactate and the glucose tissue signals were found to primarily discriminate the different histological samples. We followed the biochemical changes of human hepatic lesions through primary (HCC) and secondary (metastases from colorectal carcinoma) liver tumors, cirrhotic tissues, and non-cirrhotic histologically-confirmed normal liver tissues adjacent to metastases, achieving a metabolic differentiation of the various pathological states based upon the variation of the intracellular lactate/glucose ratio. It is suggested that such a signal pattern may act as a potential marker for assessing pathological hepatic lesions.     

The Genome Sequence of Psychrobacter arcticus 273-4, a Psychroactive Siberian Permafrost Bacterium,Reveals Mechanisms for Adaptation to Low-Temperature Growth

Psychrobacter arcticus strain 273-4, which grows at temperatures as low as −10°C, is the first cold-adapted bacterium from a terrestrial environment whose genome was sequenced. Analysis of the 2.65-Mb genome suggested that some of the strategies employed by P. arcticus 273-4 for survival under cold and stress conditions are changes in membrane composition, synthesis of cold shock proteins, and the use of acetate as an energy source. Comparative genome analysis indicated that in a significant portion of the P. arcticus proteome there is reduced use of the acidic amino acids and proline and arginine, which is consistent with increased protein flexibility at low temperatures. Differential amino acid usage occurred in all gene categories, but it was more common in gene categories essential for cell growth and reproduction, suggesting that P. arcticus evolved to grow at low temperatures. Amino acid adaptations and the gene content likely evolved in response to the long-term freezing temperatures (−10°C to −12°C) of the Kolyma (Siberia) permafrost soil from which this strain was isolated. Intracellular water likely does not freeze at these in situ temperatures, which allows P. arcticus to live at subzero temperatures.Temperature is one of the most important parameters that determine the distribution and extent of life on earth, and it does this by affecting cell structure and function. High temperatures break covalent bonds and ionic interactions between molecules, inactivating proteins and disrupting cell structures. Low temperatures reduce biochemical reaction rates and substrate transport and induce the formation of ice that damages cell structures. Not surprisingly, an organism''s compatibility with the temperature of its habitat is ultimately determined by its underlying genetic architecture.The strong emphasis in research on mesophile biology (temperatures in the 20°C to 37°C range) has given us a misimpression of the importance of cold on earth. However, 70% of the Earth''s surface is covered by oceans with average temperatures between 1°C and 5°C (11), 20% of the Earth''s terrestrial surface is permafrost (47), and a larger portion of the surface undergoes seasonal freezing, making our planet a predominantly cold environment. Hence, cold adaptation in the microbial world should be expected (55).Permafrost is defined as soils or sediments that are continuously exposed to a temperature of 0°C or less for at least 2 years (44). Permafrost temperatures range from −10°C to −20°C in the Arctic and from −10°C to −65°C in the Antarctic, and permafrost has low water activity, often contains small amounts of carbon (0.85 to 1%), and is subjected to prolonged exposure to damaging gamma radiation from ⁴⁰K in soil minerals (49). Liquid water occurs as a very thin, salty layer surrounding the soil particles in the frozen layer. Despite the challenges of the permafrost, a variety of microorganisms successfully colonize this environment, and many microorganisms have been isolated from it (54, 70). The bacterial taxa most frequently isolated from the Kolyma permafrost of northeast Siberia include Arthrobacter, Exiguobacterium, Flavobacterium, Sphingomonas, and Psychrobacter (71). Rhode and Price (56) proposed that microorganisms can survive in frozen ice for very long periods due to the very thin film of water surrounding each cell that serves as a reserve of substrates. Permafrost is a more favorable environment than ice as a result of its heterogeneous soil particles and larger reservoirs of nutrients.The genus Psychrobacter comprises a group of Gram-negative, rod-shaped, heterotrophic bacteria, and many Psychrobacter species are capable of growth at low temperatures. Members of this genus can grow at temperatures between −10°C and 42°C, and they have frequently been isolated from various cold environments, including Antarctic sea ice, ornithogenic soil and sediments, the stomach contents of Antarctic krill (Euphausia), deep seawater, and permafrost (9, 36, 57, 70, 71, 76; http://www.bacterio.cict.fr/p/psychrobacter.html). Psychrobacter arcticus 273-4 is a recently described species (4) that was isolated from a 20,000- to 30,000-year-old continuously frozen permafrost horizon in the Kolyma region in Siberia that was not exposed to temperatures higher than 4°C during isolation (70). This strain, the type strain of the species, grows at temperatures ranging from −10°C to 28°C, has a generation time of 3.5 days at −2.5°C, exhibits excellent long-term survival under freezing conditions, and has temperature-dependent physiological modifications in membrane composition and carbon source utilization (50). The fact that Psychrobacter has been found to be an indicator genus for permafrost and other polar environments (66) suggests that many of its members are adapted to low temperatures and increased levels of osmotica and have evolved molecular-level changes that aid survival at low temperatures.Early studies on cold adaptation in microorganisms revealed physiological strategies to deal with low temperatures, such as changes in membrane saturation, accumulation of compatible solutes, and the presence of cold shock proteins (CSPs) and many other proteins with general functions (62). However, many of the studies were conducted with mesophilic microorganisms, which limits the generality of the conclusions. We addressed the question of cold adaptation by studying microorganisms isolated from subzero environments using physiologic and genomic methods. We chose P. arcticus as our model because of its growth at subzero temperatures and widespread prevalence in permafrost. This paper focuses on the more novel potential adaptations.     

Characterization of mature rat oligodendrocytes: a proteomic approach

Oligodendrocytes are glial cells responsible for the synthesis and maintenance of myelin in the central nervous system (CNS). Oligodendrocytes are vulnerable to damage occurring in a variety of neurological diseases. Understanding oligodendrocyte biology is crucial for the dissemination of de- and remyelination mechanisms. The goal of the present study is the construction of a protein database of mature rat oligodendrocytes. Post-mitotic oligodendrocytes were isolated from mature Wistar rats and subjected to immunocytochemistry. Proteins were extracted and analyzed by means of two-dimensional gel electrophoresis and two-dimensional liquid chromatography, both coupled to mass spectrometry. The combination of the gel-based and gel-free approach resulted in confident identification of a total of 200 proteins. A minority of proteins were identified in both proteomic strategies. The identified proteins represent a variety of functional groups, including novel oligodendrocyte proteins. The results of this study emphasize the power of the applied proteomic strategy to study known or to reveal new proteins and to investigate their regulation in oligodendrocytes in different disease models.     

The role of WWP1-Gag interaction and Gag ubiquitination in assembly and release of human T-cell leukemia virus type 1

The PPPY motif in the matrix (MA) domain of human T-cell leukemia virus type 1 (HTLV-1) Gag associates with WWP1, a member of the HECT domain containing family of E3 ubiquitin ligases. Mutation of the PPPY motif arrests particle assembly at an early stage and abolishes ubiquitination of MA. Similar effects are seen when Gag is expressed in the presence of a truncated form of WWP1 that lacks the catalytically active HECT domain (C2WW). To understand the role of ubiquitination in budding, we mutated the four lysines in MA to arginines and identified lysine 74 as the unique site of ubiquitination. Virus-like particles produced by the K74R mutant did not contain ubiquitinated MA and showed a fourfold reduction in the release of infectious particles. Furthermore, the K74R mutation rendered assembly hypersensitive to C2WW inhibition; K74R Gag budding was inhibited at significantly lower levels of expression of C2WW compared with wild-type Gag. This finding indicates that the interaction between Gag and WWP1 is required for functions other than Gag ubiquitination. Additionally, we show that the PPPY⁻ mutant Gag exerts a strong dominant-negative effect on the budding of wild-type Gag, further supporting the importance of recruitment of WWP1 to achieve particle assembly.     

Novel fluorescent sensing system for alpha-fructosyl amino acids based on engineered fructosyl amino acid binding protein

A novel fluorescent sensing system for alpha-glycated amino acids was created based on fructosyl amino acid binding protein (FABP) from Agrobacterium tumefaciens. The protein was found to bind specifically to the alpha-glycated amino acids fructosyl glutamine (Fru-Gln) and fructosyl valine (Fru-Val) while not binding to epsilon-fructosyl lysine. An Ile166Cys mutant of FABP was created by genetic engineering and modified with the environmentally sensitive fluorophore acrylodan. The acrylodan-conjugated mutant FABP showed eight-fold greater sensitivity to Fru-Val than the unconjugated protein and could detect concentrations as low as 17 nM, making it over 100-fold more sensitive than enzyme-based detection systems. Its high sensitivity and specificity for alpha-substituted fructosyl amino acids makes the new sensing system ideally suited for the measurement of hemoglobin A1c (HbA1c), a major marker of diabetes.     

Cross-presentation of caspase-cleaved apoptotic self antigens in HIV infection

We found that the proteome of apoptotic T cells includes prominent fragments of cellular proteins generated by caspases and that a high proportion of distinct T cell epitopes in these fragments is recognized by CD8+ T cells during HIV infection. The frequencies of effector CD8+ T cells that are specific for apoptosis-dependent epitopes correlate with the frequency of circulating apoptotic CD4+ T cells in HIV-1-infected individuals. We propose that these self-reactive effector CD8+ T cells may contribute to the systemic immune activation during chronic HIV infection. The caspase-dependent cleavage of proteins associated with apoptotic cells has a key role in the induction of self-reactive CD8+ T cell responses, as the caspase-cleaved fragments are efficiently targeted to the processing machinery and are cross-presented by dendritic cells. These findings demonstrate a previously undescribed role for caspases in immunopathology.     

Biofilm formation by asymptomatic and virulent urinary tract infectious Escherichia coli strains

Escherichia coli is the most common organism associated with asymptomatic bacteriuria (ABU) in humans. In contrast to uropathogenic E. coli (UPEC) that cause symptomatic urinary tract infection, very little is known about the mechanisms by which these strains colonize the urinary tract. Here, we have investigated the biofilm-forming capacity on abiotic surfaces of groups of ABU strains and UPEC strains in human urine. We found that there is a strong bias; ABU strains were significantly better biofilm formers than UPEC strains. Our data suggest that biofilm formation in urinary tract infectious E. coli seems to be associated with ABU strains and appears to be an important strategy used by these strains for persistence in this high-flow environment.     

Endogenous retroviral insertion in Cryge in the mouse No3 cataract mutant

No3 (nuclear opacity 3) is a novel congenital nuclear cataract in mice. Microsatellite mapping placed the No3 locus on chromosome 1 between D1Mit480 (32cM) and D1Mit7 (41cM), a region containing seven crystallin genes; Cryba2 and the Cryga-Crygf cluster. Although polymorphic variants were observed, no candidate mutations were found for six of the genes. However, DNA walking identified a murine endogenous retrovirus (IAPLTR1: ERVK) insertion in exon 3 of Cryge, disrupting the coding sequence for gammaE-crystallin. Recombinant protein for the mutant gammaE was completely insoluble. The No3 cataract is mild compared with the effects of similar mutations of gammaE. Quantitative RT-PCR showed that gammaE/F mRNA levels are reduced in No3, suggesting that the relatively mild phenotype results from suppression of gammaE levels due to ERVK insertion. However, the severity of cataract is also strain dependent suggesting that genetic background modifiers also play a role in the development of opacity.