Canonical Wnt signaling: high-throughput RNAi widens the path

The canonical Wnt signaling pathway is highly conserved in evolution, widely used throughout animal development, and frequently hyperactive in cancer. Although Wnt signaling has been the subject of extensive genetic analysis in the past, some 200 genes have now been identified as candidate modulators of this pathway by a recent study using high-throughput RNAi screening.     

Molecular phylogenetics at the population/species interface in cave spiders of the southern Appalachians (Araneae:Nesticidae:Nesticus)

This paper focuses on the relationship between population genetic structure and speciation mechanisms in a monophyletic species group of Appalachian cave spiders (Nesticus). Using mtDNA sequence data gathered from 256 individuals, I analyzed patterns of genetic variation within and between populations for three pairs of closely related sister species. Each sister-pair comparison involves taxa with differing distributional and ecological attributes; if these ecological attributes are reflected in basic demographic differences, then speciation might proceed differently across these sister taxa comparisons. Both frequency-based and gene tree analyses reveal that the genetic structure of the Nesticus species studied is characterized by similar and essentially complete population subdivision, regardless of differences in general ecology. These findings contrast with results of prior genetic studies of cave-dwelling arthropods that have typically revealed variation in population structure corresponding to differences in general ecology. Species fragmentation through both extrinsic and intrinsic evolutionary forces has resulted in discrete, perhaps independent, populations within morphologically defined species. Large sequence divergence values observed between populations suggest that this independence may extend well into the past. These patterns of mtDNA genealogical structure and divergence imply that species as morphological lineages are currently more inclusive than basal evolutionary or phylogenetic units, a suggestion that has important implications for the study of speciation mechanisms.      

Chronic stress and its effects on adrenal cortex apoptosis in pregnant rats

The model of chronic intermittent stress by immobilization during pregnancy may produce alterations in the mechanisms that maintain adrenal gland homeostasis. In earlier investigations using this model, significant variations in plasma prolactin and corticosterone levels, and adrenal gland weights were observed. We hypothesized that chronic stress causes changes in apoptosis in the adrenal glands of pregnant rats. We identified and quantified apoptotic cells in the adrenal cortex and examined their ultrastructural characteristics using transmission electron microscopy. Adrenal glands of pregnant rats at gestation days 12, 17 and 21 were studied for control and experimental (stressed) rats. Immunolabelling techniques, stereological analysis and image quantification of adrenal gland sections were combined to determine differences in apoptosis in the different cell populations of the adrenal cortex. The apoptotic index of the experimental rats showed a significant reduction at gestation day 17, while at days 12 and 21 there were no differences from controls. Moreover, the apoptotic index of the reticular zones in control and experimental animals showed a significant increase compared to the glomerular and fascicular zones at the three gestation times studied. Chronic stress by immobilization reduced the caspase-dependent apoptotic index at gestation day 17, which may be related to variations in plasma concentrations of estrogens and prolactin.     

HistoChoice® as an alternative to formalin fixation of undecalcified bone specimens

We compared histochemical and immunohistochemical staining as well as fluorochrome labeling in murine bone specimens that were fixed with 10% neutral buffered formalin to those fixed with HistoChoice®. We showed that sections from undecalcified tibiae fixed for 4 h in HistoChoice® resulted in enhanced toluidine blue and Von Kossa histochemical staining compared to formalin fixation. HistoChoice® produced comparable or improved staining for alkaline phosphatase. Acid phosphatase localization was better in formalin fixed specimens, but osteoclasts were visuralized more easily in HistoChoice® fixed specimens. As expected, immunohistochemical labeling was antibody dependent; some antibodies labeled better in HistoChoice® fixed specimens while others were better in formalin fixed specimens. Toluidine blue, Von Kossa, and alkaline phosphatase staining of sections fixed for 12 h produced sections that were similar to 4 h fixed sections. Fixation for 12 h preserved acid phosphatase activity better. Increasing fixation to 12 h affected immunolocalization differentially. Bone sialoprotein labeling in HistoChoice® fixed specimens was comparable to formalin fixed samples. On the other hand, after 12 h formalin fixation, osteocalcin labeling was comparable to HistoChoice®. For most histochemical applications, fixing murine bone specimens for 4 h with HistoChoice® yielded superior staining compared to formalin fixation. If immunohistochemical localization is desired, however, individual antibodies must be tested to determine which fixation process retains antigenicity better. In addition, there was no detectable difference in the intensity of fluorochrome labeling using either fixative. Finally, fixation duration did not alter the intensity of labeling.     

Development of sequence amplified characterized region (SCAR) markers of helicoverpa armigera: a new polymerase chain reaction-based technique for predator gut analysis

A method is described for the development of DNA markers for detection of Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae) in predator gut analysis, based on sequence characterized amplified regions (SCARs) derived from a randomly amplified polymorphic DNA (RAPD) band. A 1200-bp DNA fragment of H. armigera, absent in the predator band pattern and in other closely related prey species, was identified by RAPD analysis. This fragment was cloned and its extremes sequenced to design extended strand-specific 20-mer oligonucleotide primers. Three pairs of SCAR primers, which amplified three different DNA fragments, were used to study the effect of fragment length on detection of prey in the predator gut. Using the pair of primers that amplified the longest fragment of H. armigera DNA, a single band of 1100 bp was obtained, but its detection was not possible in the predator gut. Detection of the ingested prey was possible with the other two pairs of SCAR primers, obtaining bands of 600 and 254 bp, respectively. Detection of H. armigera DNA in the gut of the predator Dicyphus tamaninii was evaluated immediately after ingestion (t = 0) and after 4 h. Detection of H. armigera DNA after 4 h was only possible using the pair of primers that amplified the shortest fragment (254 bp). The test for specificity, using these last pair of primers, showed that H. armigera was the only species detected. The detection threshold was defined at a 1:8192 dilution of a H. armigera whole egg in all samples.     

Trimetazidine improves left ventricular function in diabetic patients with coronary artery disease: a double-blind placebo-controlled study

Background

Patients with diabetic cardiomyopathy have an impaired myocardial glucose handling and distal distribution of coronary atherosclerosis. Trimetazidine, an anti-ischemic metabolic agent, improves myocardial glucose utilization though inhibition of fatty acid oxidation. Aim of the present study was to evaluate whether the metabolic effect of trimetazidine on left ventricular function in patients with diabetic cardiomyopathy.

Methods

32 patients (24 males and 8 females, mean (SE) age = 67 ± 6 years) with type 2 diabetes and ischemic cardiomyopathy were randomized to receive either trimetazidine (20 mg, t.d.s.) or placebo (t.d.s.) for six months in a randomized parallel study. Patients performed an echocardiogram at baseline and after 6 months.

Results

Demographic data were comparable between the two groups. After six month baseline left ventricular end-diastolic diameters increased from 62.4 ± 1.7 to 63 ± 2.1 mm in the placebo group, while decreased from 63.2 ± 2.1 to 58 ± 1.6 mm (p < 0.01 compared to baseline) in the trimetazidine group. Compared to baseline, left ventricular ejection fraction increased by 5.4 ± 0.5% (p < 0.05) in the trimetazidine group while remained unchanged in the placebo group -2.4 ± 1.1% (NS), p < 0.01 between groups. A significant improvement in wall motion score index and in the E/A wave ratio was detected in patients treated with trimetazidine, but not in those receiving placebo.

Conclusion

in diabetic patients with ischemic heart disease trimetazidine added to standard medical therapy has beneficial effect on left ventricular volumes and on left ventricular ejection fraction compared to placebo. This effect may be related to the effect of trimetazidine upon cardiac glucose utilization.     

Cell cycle -dependent proteolysis in plants. Identification Of the destruction box pathway and metaphase arrest produced by the proteasome inhibitor mg132

It is widely assumed that mitotic cyclins are rapidly degraded during anaphase, leading to the inactivation of the cell cycle-dependent protein kinase Cdc2 and allowing exit from mitosis. The proteolysis of mitotic cyclins is ubiquitin/26S proteasome mediated and requires the presence of the destruction box motif at the N terminus of the proteins. As a first attempt to study cyclin proteolysis during the plant cell cycle, we investigated the stability of fusion proteins in which the N-terminal domains of an A-type and a B-type tobacco mitotic cyclin were fused in frame with the chloramphenicol acetyltransferase (CAT ) reporter gene and constitutively expressed in transformed tobacco BY2 cells. For both cyclin types, the N-terminal domains led the chimeric cyclin-CAT fusion proteins to oscillate in a cell cycle-specific manner. Mutations within the destruction box abolished cell cycle-specific proteolysis. Although both fusion proteins were degraded after metaphase, cyclin A-CAT proteolysis was turned off during S phase, whereas that of cyclin B-CAT was turned off only during the late G2 phase. Thus, we demonstrated that mitotic cyclins in plants are subjected to post-translational control (e.g., proteolysis). Moreover, we showed that the proteasome inhibitor MG132 blocks BY2 cells during metaphase in a reversible way. During this mitotic arrest, both cyclin-CAT fusion proteins remained stable.     

Role of calcium in signal transduction during the hypersensitive response caused by basidiospore-derived infection of the cowpea rust fungus

The hypersensitive response (HR) of disease-resistant plant cells to fungal invasion is a rapid cell death that has some features in common with programmed cell death (apoptosis) in animals. We investigated the role of cytosolic free calcium ([Ca2+]i) in the HR of cowpea to the cowpea rust fungus. By using confocal laser scanning microscopy in conjunction with a calcium reporter dye, we found a slow, prolonged elevation of [Ca2+]i in epidermal cells of resistant but not susceptible plants as the fungus grew through the cell wall. [Ca2+]i levels declined to normal levels as the fungus entered and grew within the cell lumen. This elevation was related to the stage of fungal growth and not to the speed of initiation of subsequent cell death. Elevated [Ca2+]i levels also represent the first sign of the HR detectable in this cowpea-cowpea rust fungus system. The increase in [Ca2+]i was prevented by calcium channnel inhibitors. This effect was consistent with pharmacological tests in which these inhibitors delayed the HR. The data suggest that elevation of [Ca2+]i is involved in signal transduction leading to the HR during rust fungal infection.     

Minimal aerobic sludge retention time in biological phosphorus removal systems

The methodology for determination of the minimally required aerobic sludge retention time (SRTminaer) in biological phosphorus removal (BPR) systems is presented in this article. Contrary to normal biological conversions, the BPR process is not limited by a SRTmin resulting from the maximum growth rate of the organisms. This is because the aerobic SRT should be long enough to oxidize the amount of poly-hydroxy-alkanoates (PHA) stored in the anaerobic phase. This means that the SRTminaer will primarily depend on the PHA conversion kinetics and the maximal achievable PHA content in the cell (storage capacity). The model for the prediction of the minimally required aerobic SRT as a function of kinetic and process parameters was developed and compared with experimental data used to evaluate several operational aspects of BPR in a sequencing batch reactor (SBR) system. The model was proved as capable of describing them satisfactorily.Copyright 1998 John Wiley & Sons, Inc.