Genome-wide MicroRNA Expression Profiles in COPD: Early Predictors for Cancer Development

Chronic obstructive pulmonary disease(COPD) significantly increases the risk of developing cancer. Biomarker studies frequently follow a case-control set-up in which patients diagnosed with a disease are compared to controls. Longitudinal cohort studies such as the COPD-centered German COPD and SYstemic consequences-COmorbidities NETwork(COSYCONET) study provide the patient and biomaterial base for discovering predictive molecular markers. We asked whether microRNA(miRNA) profiles in blood collected from COPD patients prior to a tumor diagnosis could support an early diagnosis of tumor development independent of the tumor type. From 2741 participants of COSYCONET diagnosed with COPD, we selected 534 individuals including 33 patients who developed cancer during the follow-up period of 54 months and 501 patients who did not develop cancer, but had similar age, gender and smoking history. Genome-wide miRNA profiles were generated and evaluated using machine learning techniques. For patients developing cancer we identified nine miRNAs with significantly decreased abundance(two-tailed unpaired t-test adjusted for multiple testing P 0.05), including members of the miR-320 family. The identified mi RNAs regulate different cancer-related pathways including the MAPK pathway(P = 2.3 x 10~(-5)). We also observed the impact of confounding factors on the generated miRNA profiles, underlining the value of our matched analysis. For selected miRNAs, qRT-PCR analysis was applied to validate the results. In conclusion, we identified several miRNAs in blood of COPD patients, which could serve as candidates for biomarkers to help identify COPD patients at risk of developing cancer.     

Dysregulation of the calpain-calpastatin system plays a role in the development of cerulein-induced acute pancreatitis in the rat

Calpain, a calcium-dependent cytosolic cysteine protease, is implicated in a multitude of cellular functions but also plays a role in cell death. Recently, we have shown that two ubiquitous isoforms, termed micro-calpain and m-calpain, are expressed in rat pancreatic acinar cells and that calcium ionophore-induced calpain activation leads to acinar cell injury. On the basis of these observations, we have now investigated the role of both calpain forms and the endogenous calpain inhibitor calpastatin in acute pancreatitis. After treatment of rats either without or with calpain inhibitor Z-Val-Phe methyl ester (ZVP; 60 mg/kg i.p.), pancreatitis was induced by cerulein injections (10 microg/kg i.p.; 5 times at hourly intervals). Calpain activation and calpastatin expression in the pancreatic tissue were studied by Western blot analysis. Pancreatic injury was assessed by plasma amylase activity, pancreatic wet/dry weight ratio (edema), histological and electron-microscopic analyses, as well as fluorescence labeling of actin filaments. Cerulein caused an activation of both micro-calpain and m-calpain, accompanied by degradation of calpastatin. Prophylactic administration of ZVP reduced the cerulein-induced calpain activation but had no effect on calpastatin alterations. In correlation to the diminished calpain activity, the severity of pancreatitis decreased as indicated by a decline in amylase activity (P < 0.01), pancreatic edema formation (P < 0.05), histological score for eight parameters (P < 0.01), and actin filament alterations. Our findings support the hypothesis that dysregulation of the calpain-calpastatin system may play a role in the onset of acute pancreatitis.     

Semen cryopreservation and the conservation of endangered species

Currently, more than 16,000 plant and animal species are imminently threatened by extinction, often as a direct consequence of anthropogenic influences. One of the measures to halt that process is genetic resource banking. This short review focuses on mammal sperm cryopreservation in combination with assisted reproduction techniques. It summarizes general problems, recent developments, and currently applied protocols and gives an overview of hitherto existing successes of assisted reproduction measures in wild animals in the light of conservation efforts.     

Phylogenetic analysis of Syntrophobacter wolinii reveals a relationship with sulfate-reducing bacteria

A 16S rRNA sequence analysis of Syntrophobacter wolinii was done by using PCR amplification of the 16S rRNA-genes from DNA isolated from the S. wolinii-Desulfovibrio sp. coculture. Phylogenetic analysis using the obtained sequence revealed that S. wolinii was not related to bacteria growing syntrophically on other fatty acids than propionate, but was related to sulfate-reducing bacteria. The closest related bacteria are Desulfomonile tiedjei and Desulfoarculus baarsii.     

Concurrent Neutral Evolution of mRNA Secondary Structures and Encoded Proteins

Messenger RNA sequences often have to preserve functional secondary structure elements in addition to coding for proteins. We present a statistical analysis of retroviral mRNA which supports the hypothesis that the natural genetic code is adapted to such complementary coding. These sequences are still able to explore efficiently the space of possible proteins by point mutations. This is borne out by the observation that, in stem regions of retroviral mRNA foldings, silent mutations on one strand are preferentially accompanied by conservative mutations on the other. Distances between amino acids based on physicochemical properties are used to quantify the conservation of protein function under the constraint of maintained RNA secondary structure. We find that preservation of RNA secondary structure by compensatory mutations is evolutionary compatible with the efficient search for new variants on the protein level. Received: 4 June 1999 / Accepted: 12 October 1999     

Complete oxidation of toluene under strictly anoxic conditions by a new sulfate-reducing bacterium.

A toluene-degrading sulfate-reducing bacterium, strain Tol2, was isolated from marine sediment under strictly anoxic conditions. Toluene was toxic if applied directly to the medium at concentrations higher than 0.5 mM. To provide toluene continuously at a nontoxic concentration, it was supplied in an inert hydrophobic carrier phase. The isolate had oval, sometimes motile cells (1.2 to 1.4 by 1.2 to 2.0 microns). The doubling time was 27 h. Toluene was completely oxidized to CO2, as demonstrated by measurement of the degradation balance. The presence of carbon monoxide dehydrogenase and formate dehydrogenase indicated a terminal oxidation of acetyl coenzyme A via the CO dehydrogenase pathway. The use of hypothetical intermediates of toluene degradation was tested in growth experiments and adaptation studies with dense cell suspensions. Results do not support a degradation of toluene via one of the cresols or methylbenzoates, benzyl alcohol, or phenylacetate as free intermediate. Benzyl alcohol did not serve as growth substrate; moreover, it was a strong, specific inhibitor of toluene degradation, whereas benzoate utilization was not affected by benzyl alcohol. Sequencing of 16S rRNA revealed a relationship to the metabolically dissimilar genus Desulfobacter and on a deeper level to the genus Desulfobacterium. The new genus and species Desulfobacula toluolica is proposed.     

Suppression of Kinetic AMP Cooperativity of Fructose-1,6-Bisphosphatase by Carbamoylation of Lysine 50

Selective treatment of pig kidney fructose 1,6-bisphosphatase with cyanate leads to the formation of an active carbamoylated derivative that shows no cooperative interaction between the AMP-binding sites, but completely retains the sensitivity to the inhibitor. By an exhaustive carbamoylation of the enzyme a derivative is formed that has a complete loss of cooperativity and a decrease of sensitivity to AMP. It was proposed that the observed changes of allosteric properties were due to the chemical modification of two lysine residues per enzyme subunit [Slebe et al. (1983), J. Protein Chem. 2, 437–443]. Studies of the temperature dependence of AMP sensitivity and the interaction with Cibacron Blue Sepharose of carbamoylated fructose 1,6-bisphosphatase derivatives indicate that the lysine residue involved in AMP sensitivity is located at the allosteric AMP site, while the lysine residue involved in AMP cooperativity is at a distinct location. Using [¹⁴C]cyanate, we identified both lysine residues in the primary structure of the enzyme; Lys50 is essential for AMP cooperativity and Lys112 appears to be the reactive residue involved in the AMP sensitivity. According to the fructose 1,6-bisphosphatase crystal structure, Lys50 is strategically positioned at the C1–C2 interface, near the molecular center of the tetramer, and Lys112 is in the AMP-binding site. The results reported here, combined with the structural data of the enzyme, strongly suggest that the C1ndash;C2 interface is critical for the propagation of the allosteric signal among the AMP sites on different subunits.