Diagnostic utility of snail in metaplastic breast carcinoma

Aims

As one of the five Lactate dehydrogenase (LDH) isoenzymes, LDH5 has the highest efficiency to catalyze pyruvate transformation to lactate. LDH5 overexpression in cancer cells induces an upregulated glycolytic metabolism and reduced dependence on the presence of oxygen. Here we analyzed LDH5 protein expression in a well characterized large cohort of primary lung cancers in correlation to clinico-pathological data and its possible impact on patient survival.

Methods

Primary lung cancers (n = 269) and non neoplastic lung tissue (n = 35) were tested for LDH5 expression by immunohistochemistry using a polyclonal LDH5 antibody (ab53010). The results of LDH5 expression were correlated to clinico-pathological data as well as to patient's survival. In addition, the results of the previously tested Transketolase like 1 protein (TKTL1) expression were correlated to LDH5 expression.

Results

89.5% (n = 238) of NSCLC revealed LDH5 expression whereas LDH5 expression was not detected in non neoplastic lung tissues (n = 34) (p < 0.0001). LDH5 overexpression was associated with histological type (adenocarcinoma = 57%, squamous cell carcinoma = 45%, large cell carcinoma = 46%, p = 0.006). No significant correlation could be detected with regard to TNM-stage, grading or survival. A two sided correlation between the expression of TKTL1 and LDH5 could be shown (p = 0.002) within the overall cohort as well as for each grading and pN group. A significant correlation between LDH5 and TKTL1 within each histologic tumortype could not be revealed.

Conclusions

LDH5 is overexpressed in NSCLC and could hence serve as an additional marker for malignancy. Furthermore, LDH5 correlates positively with the prognostic marker TKTL1. Our results confirm a close link between the two metabolic enzymes and indicate an alteration in the glucose metabolism in the process of malignant transformation.     

The role of matrix metalloproteinases 2 and 9 during rat tongue carcinogenesis induced by 4-nitroquinoline 1-oxide

Matrix metalloproteinases (MMPs) are implicated in a wide range of physiological and pathological processes, including morphogenesis, wound healing, angiogenesis, inflammation, and cancer. The purpose of this study was to characterize the role of MMPs as depicted by the expression of MMP-2 and MMP-9 during 4-nitroquinoline 1-oxide-induced rat tongue carcinogenesis. Male Wistar rats were distributed into three groups of 10 animals each and treated with 4-nitroquinoline 1-oxide solution at 50 ppm through their drinking water for 4, 12, and 20 weeks. Ten animals were used as control group. No histopathological abnormalities were induced in the epithelium after 4 weeks of carcinogen exposure; however, immunoexpression of MMP-2 was noticed. The same picture occurred to MMP-9, in which positive expression was detected for this immunomarker. MMP-2 and MMP-9 showed positive expression either in pre-neoplastic lesions at 12 weeks following carcinogen exposure or in well-differentiated squamous cell carcinoma induced after 20 weeks of treatment with 4NQO. Taken together, our results support the belief that MMP-2 and MMP-9 play important role during malignant transformation and conversion of oral mucosa as assessed by immunohistochemistry.     

Cg2091 encodes a polyphosphate/ATP-dependent glucokinase of Corynebacterium glutamicum

The Corynebacterium glutamicum gene cg2091 is encoding a polyphosphate (PolyP)/ATP-dependent glucokinase (PPGK). Previous work demonstrated the association of PPGK to PolyP granules. The deduced amino acid sequence of PPGK shows 45% sequence identity to PolyP/ATP glucomannokinase of Arthrobacter sp. strain KM and 50% sequence identity to PolyP glucokinase of Mycobacterium tuberculosis H37Rv. PPGK from C. glutamicum was purified from recombinant Escherichia coli. PolyP was highly preferred over ATP and other NTPs as substrate and with respect to the tested PolyPs differing in chain length; the protein was most active with PolyP₇₅. Gel filtration analysis revealed that PolyP supported the formation of homodimers of PPGK and that PPGK was active as a homodimer. A ppgK deletion mutant (ΔppgK) showed slowed growth in minimal medium with maltose as sole carbon source. Moreover, in minimal medium containing 2 to 4% (w/v) glucose as carbon source, ΔppgK grew to lower final biomass concentrations than the wild type. Under phosphate starvation conditions, growth of ΔppgK was reduced, and growth of a ppgK overexpressing strain was increased as compared to wild type and empty vector control, respectively. Thus, under conditions of glucose excess, the presence of PPGK entailed a growth advantage.     

Molecular machinery of mitochondrial dynamics in yeast

Mitochondria are amazingly dynamic organelles. They continuously move along cytoskeletal tracks and frequently fuse and divide. These processes are important for maintenance of mitochondrial functions, for inheritance of the organelles upon cell division, for cellular differentiation and for apoptosis. As the machinery of mitochondrial behavior has been highly conserved during evolution, it can be studied in simple model organisms, such as yeast. During the past decade, several key components of mitochondrial dynamics have been identified and functionally characterized in Saccharomyces cerevisiae. These include the mitochondrial fusion and fission machineries and proteins required for maintenance of tubular shape and mitochondrial motility. Taken together, these findings reveal a comprehensive picture that shows the cellular processes and molecular components required for mitochondrial inheritance and morphogenesis in a simple eukaryotic cell.     

Calcium uptake in mosses and its role in stone biodeterioration

The biodeterioration of stone substrata by bryophytes (Musci and Hepaticae) is due to biogeochemical and biogeophysical mechanisms. In this work the biogeochemical damage caused by epilithic moss species, found in archaeological sites of Rome, was investigated. The cellular calcium content in specimens of Grimmia pulvinata (Hedw.) Sm. was analysed using a sequential elution technique. The analyses were carried out on moss specimens sampled from different lithotypes and in different seasons in order to measure the calcium concentration both in relation to the mineral composition of the stone and to the plant physiology. The highest calcium concentration of the extracellular exchangeable fraction was detected in the samples grown on marble and in the waterlogged specimen sampled in spring time.     

Glutathione cycle impairment mediates A beta-induced cell toxicity

Alzheimer's disease is widely held to be associated with oxidative stress due, in part, to the action of amyloid beta-peptide (A beta). We observed that A beta 25-35 induced an increase in reactive oxygen species (ROS) in NT2 rho+ cells, leading to protein and lipid oxidation. This oxidative status was partially prevented by the antioxidants, vitamin E, reduced glutathione, and by melatonin. However, NT2 rho0 cells (that lack mitochondrial DNA) in the absence of A beta showed an increase in ROS production, lipid and protein oxidation, as compared with parental rho+ cells. Upon A beta 25-35 treatment, in rho+ cells, a decrease in glutathione reductase activity and in GSH levels was observed, whereas glutathione peroxidase activity was shown to be increased. In NT2 rho0 cells, in the absence of A beta, GSH levels were maintained, whereas glutathione reductase and peroxidase activities were increased. The exposure of A beta to rho0 cells did not induce any change in these parameters. We observed that melatonin prevented caspase activation and DNA fragmentation in rho+ cells treated with A beta. Considering the evidence presented, we argue that the glutathione cycle impairment is a key event in A beta-induced cell toxicity.     

Mycorrhizal colonization mediated by species interactions in arctic tundra

The Alaskan tussock tundra is a strongly nutrient-limited ecosystem, where almost all vascular plant species are mycorrhizal. We established a long-term removal experiment to document effects of arctic plant species on ecto- and ericoid mycorrhizal fungi and to investigate whether species interactions and/or nutrient availability affect mycorrhizal colonization. The treatments applied were removal of Betula nana (Betulaceae, dominant deciduous shrub species), removal of Ledum palustre (Ericaceae, dominant evergreen shrub species), control (no removal), and each of these three treatments with the addition of fertilizer. After 3 years of Ledum removal and fertilization, we found that overall ectomycorrhizal colonization in Betula was significantly reduced. Changes in ectomycorrhizal morphotype composition in removal and fertilized treatments were also observed. These results suggest that the effect of Ledum on Betula 's mycorrhizal roots is due to sequestration of nutrients by Ledum, leading to reduced nutrient availability in the soil. In contrast, ericoid mycorrhizal colonization was not affected by fertilization, but the removal of Betula and to a lower degree of Ledum resulted in a reduction of ericoid mycorrhizal colonization suggesting a direct effect of these species on ericoid mycorrhizal colonization. Nutrient availability was only higher in fertilized treatments, but caution should be taken with the interpretation of these data as soil microbes may effectively compete with the ion exchange resins for the nutrients released by plant removal in these nutrient-limited soils.     

Real-time PCR based on SYBR-Green I fluorescence: An alternative to the TaqMan assay for a relative quantification of gene rearrangements, gene amplifications and micro gene deletions

Background

Real-time PCR is increasingly being adopted for RNA quantification and genetic analysis. At present the most popular real-time PCR assay is based on the hybridisation of a dual-labelled probe to the PCR product, and the development of a signal by loss of fluorescence quenching as PCR degrades the probe. Though this so-called 'TaqMan' approach has proved easy to optimise in practice, the dual-labelled probes are relatively expensive.

Results

We have designed a new assay based on SYBR-Green I binding that is quick, reliable, easily optimised and compares well with the published assay. Here we demonstrate its general applicability by measuring copy number in three different genetic contexts; the quantification of a gene rearrangement (T-cell receptor excision circles (TREC) in peripheral blood mononuclear cells); the detection and quantification of GLI, MYC-C and MYC-N gene amplification in cell lines and cancer biopsies; and detection of deletions in the OPA1 gene in dominant optic atrophy.

Conclusion

Our assay has important clinical applications, providing accurate diagnostic results in less time, from less biopsy material and at less cost than assays currently employed such as FISH or Southern blotting.     

Changing the conformation state of cytochrome b558 initiates NADPH oxidase activation: MRP8/MRP14 regulation

Phagocyte NADPH oxidase generates O2. for defense mechanisms and cellular signaling. Myeloid-related proteins MRP8 and MRP14 of the S100 family are EF-hand calcium-binding proteins. MRP8 and MRP14 were co-isolated from neutrophils on an anti-p47phox matrix with oxidase cytosolic factors and identified by mass spectrometry. MRP8 and MRP14 are absent from Epstein-Barr virus-immortalized B lymphocytes, and, coincidentally, these cells display weak oxidase activity compared with neutrophils. MRP8/MRP14 that was purified from neutrophils enhanced oxidase turnover of B cells in vitro, suggesting that MRP8/MRP14 is involved in the activation process. This was confirmed ex vivo by co-transfection of Epstein-Barr virus-transformed B lymphocytes with genes encoding MRP8 and MRP14. In a semi-recombinant cell-free assay, recombinant MRP8/MRP14 increased the affinity of p67phox for cytochrome b558 synergistically with p47phox. Moreover, MRP8/MRP14 initiated oxidase activation on its own, through a calcium-dependent specific interaction with cytochrome b558 as shown by atomic force microscopy and a structure-function relationship investigation. The data suggest that the change of conformation in cytochrome b558, which initiates the electron transfer, can be mediated by effectors other than oxidase cytosolic factors p67phox and p47phox. Moreover, MRP8/MRP14 dimer behaves as a positive mediator of phagocyte NADPH oxidase regulation.