Gene-targeted mice lacking the Trex1 (DNase III) 3'-->5' DNA exonuclease develop inflammatory myocarditis

TREX1, originally designated DNase III, was isolated as a major nuclear DNA-specific 3'-->5' exonuclease that is widely distributed in both proliferating and nonproliferating mammalian tissues. The cognate cDNA shows homology to the editing subunit of the Escherichia coli replicative DNA polymerase III holoenzyme and encodes an exonuclease which was able to serve a DNA-editing function in vitro, promoting rejoining of a 3' mismatched residue in a reconstituted DNA base excision repair system. Here we report the generation of gene-targeted Trex1(-/-) mice. The null mice are viable and do not show the increase in spontaneous mutation frequency or cancer incidence that would be predicted if Trex1 served an obligatory role of editing mismatched 3' termini generated during DNA repair or DNA replication in vivo. Unexpectedly, Trex1(-/-) mice exhibit a dramatically reduced survival and develop inflammatory myocarditis leading to progressive, often dilated, cardiomyopathy and circulatory failure.     

Increased expression of H11 kinase stimulates glycogen synthesis in the heart

Mechanical forces related to pressure and flow are important for cell hypertrophy and proliferation. There are still a few studies that examine responses of human vascular smooth muscle cells to pure pressure stress (transmural pressure). It is unclear as to which mechanisms are involved in cellular responses to pressure elevation. On the other hand, although the involvement of the local renin-angiotensin system (RAS) in pressure-induced responses was reported, the results were contradictory. It still remains to be determined whether RAS in human vascular smooth muscle cells is activated by pure pressure stress. We studied the upstream signal transduction events of extracellular signal kinase (ERK) in response to atmospheric pressure stress and involvement of angiotensin II in pressure-induced cell proliferation in human aortic smooth muscle cells (HASMC). A pressure-loading apparatus was set up to examine the effects of atmospheric pressure on human aortic smooth muscle cells. Pressure application of 160 mmHg for 3 h produced cell proliferation and activated ERK and c-JUN N-terminal kinase (JNK). ACE inhibitor suppressed all of them. ERK kinase (MEK) inhibitor also suppressed cell proliferation stimulated by pure pressure. The phosphorylated c-Src was increased by pure pressure stress. The treatment with c-Src kinase inhibitor suppressed pressure-induced proliferative response. In summary, our study found that ERK activation mediated pure pressure-induced proliferative response of HASMC. This activation was partly mediated by c-Src.     

Two opposite effects of cofilin on the thermal unfolding of F-actin: a differential scanning calorimetric study

Differential scanning calorimetry was used to examine the effects of cofilin on the thermal unfolding of actin. Stoichiometric binding increases the thermal stability of both G- and F-actin but at sub-saturating concentrations cofilin destabilizes F-actin. At actin:cofilin molar ratios of 1.5-6 the peaks corresponding to stabilized (66-67 degrees C) and destabilized (56-57 degrees C) F-actin are observed simultaneously in the same thermogram. Destabilizing effects of sub-saturating cofilin are highly cooperative and are observed at actin:cofilin molar ratios as low as 100:1. These effects are abolished by the addition of phalloidin or aluminum fluoride. Conversely, at saturating concentrations, cofilin prevents the stabilizing effects of phalloidin and aluminum fluoride on the F-actin thermal unfolding. These results suggest that cofilin stabilizes those actin subunits to which it directly binds, but destabilizes F-actin with a high cooperativity in neighboring cofilin-free regions.     

Complexity of phenotypes and symbiotic behaviour of <Emphasis Type="Italic">Rhizobium leguminosarum</Emphasis> biovar <Emphasis Type="Italic">trifolii</Emphasis> exopolysaccharide mutants

Quantitative in vitro antibacterial activities, i.e., minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs), of 12 -lactam antibiotics against Agrobacterium tumefaciens strains LBA4404 and EHA101 were examined, in order to identify antibiotics effective in eliminating the bacteria in Agrobacterium-mediated plant genetic transformation. The antibacterial activities of -lactams tested against strain EHA101 were equal to or less than those tested against strain LBA4404. Cefotaxime, cefbuperazone, and meropenem had high activities against strain LBA4404 (MBC <1 mg l^–1). Against strain EHA101, however, only meropenem showed activity comparable to that against strain LBA4404. The production of -lactamase was observed only in strain EHA101.Abbreviations CFU Colony-forming unit - MBC Minimum bactericidal concentration - MIC Minimum inhibitory concentration - PBP Penicillin-binding protein     

The preferred route for the degradation of silencing target RNAs in transgenic plants depends on pre-established silencing conditions

RNA silencing can be initiated upon dsRNA accumulation and results in homology-dependent degradation of target RNAs mediated by 21–23 nt small interfering RNAs (siRNAs). These small regulatory RNAs can direct RNA degradation via different routes such as the RdRP/Dicer- and the RNA-induced silencing complex (RISC)-catalysed pathways. The relative contribution of both pathways to degradation of target RNAs is not understood. To gain further insight in the process of target selection and degradation, we analysed production of siRNAs characteristic for Dicer-mediated RNA degradation during silencing of mRNAs and chimeric viral RNAs in protoplasts from plants of a transgenic tobacco silencing model line. We show that small RNA accumulation is limited to silencing target regions during steady-state mRNA silencing. For chimeric viral RNAs, siRNA production appears dependent on pre-established cellular silencing conditions. The observed siRNA accumulation profiles imply that silencing of viral target RNAs in pre-silenced protoplasts occurs mainly via a RISC-mediated pathway, guided by (pre-existing) siRNAs derived from cellular mRNAs. In cells that are not silenced at the time of infection, viral RNA degradation seems to involve Dicer action directly on the viral RNAs. This suggests that the silencing mechanism flexibly deploys different components of the RNA degradation machinery in function of the prevailing silencing status.     

Sensitive and specific detection of microRNAs by northern blot analysis using LNA-modified oligonucleotide probes

We describe here a new method for highly efficient detection of microRNAs by northern blot analysis using LNA (locked nucleic acid)-modified oligonucleotides. In order to exploit the improved hybridization properties of LNA with their target RNA molecules, we designed several LNA-modified oligonucleotide probes for detection of different microRNAs in animals and plants. By modifying DNA oligonucleotides with LNAs using a design, in which every third nucleotide position was substituted by LNA, we could use the probes in northern blot analysis employing standard end-labelling techniques and hybridization conditions. The sensitivity in detecting mature microRNAs by northern blots was increased by at least 10-fold compared to DNA probes, while simultaneously being highly specific, as demonstrated by the use of different single and double mismatched LNA probes. Besides being highly efficient as northern probes, the same LNA-modified oligonucleotide probes would also be useful for miRNA in situ hybridization and miRNA expression profiling by LNA oligonucleotide microarrays.     

CRM197 (nontoxic diphtheria toxin): effects on advanced cancer patients

Purpose: Many years ago, diphtheria toxin (DT) showed antitumor activity in mice and in humans, but it was unclear whether this depended on the toxicity of the molecule only or on its strong inflammatory-immunological property as well. To deal with this open question, we planned to treat a group of cancer patients with cross-reacting material 197 (CRM197). CRM197 is a nontoxic mutant of DT that shares the immunological properties of the native molecule and its ability to bind to heparin-binding epidermal growth factor (HB-EGF), the specific cell-membrane receptor for DT that is often overexpressed in cancer. Methods: 25 outpatients with various advanced tumors who were refractory to standard therapies (23 subjects) or had refused, in whole or in part, conventional therapies (2 subjects) were treated with CRM197 injected subcutaneously in the abdominal wall, on alternate days, for 6 days. Three different dosages (1.7, 2.6, or 3.5 mg/day) were used according to the patients degree of immunological reactivity to DT/CRM197 (none, moderate, or high). Results: After the first administration of CRM197, a significant increase in the number of circulating neutrophils and in the serum level of TNF- was detected. Toxicities were minimal. Only patients with delayed-type hypersensitivity to DT/CRM197 had irritating skin reactions in the injection sites and a flu-like syndrome with fever. Pharmacokinetics showed a mean peak concentration (12.7 ng/ml) 12 h after the first injection and a mean half-life of 18.1 h. There were two complete and one partial responses (metastatic breast carcinoma, neuroblastoma, and metastatic breast carcinoma) lasting 4, 45+, and 15 months, respectively. Six cases of stable disease, lasting from 1 to 15 months, were also recorded. Conclusions: CRM197 injected subcutaneously elicited an inflammatory-immunological reaction, caused tolerable toxicities, was absorbed to a good extent into the circulatory system, and exerted some degree of biological antitumor activity. A possible role of neutrophils and TNF- in the mode of action of the molecule is hypothesized.     

Phase-synchronization of daily motor activities can reveal differential circadian patterns

The aim of the study was to determine any alteration of the 24 h motor activity pattern of a bipolar patient in different mood states. Actigraphic records were collected on an outpatient basis for a total of 387 days. The daily actograms were synchronized in phase to the time of morning awakening before averaging, which significantly enhanced the structure of the averaged traces. The actograms were divided into three groups based on total daily count. The daily motor activity patterns of the low- and high-activity days have a different circadian pattern. We propose it may have a relevance to the different mood states. The phase-synchronization of the 24 h actograms to the patient's sleep-wake cycle, specifically to the time of awaking from the nighttime sleep, may help reveal differences in the daily temporal patterns of motor activity.