Myometrial apoptosis: activation of the caspase cascade in the pregnant rat myometrium at midgestation

In the present study, we determined the contribution of myometrial hyperplasia, hypertrophy, and apoptosis to uterine growth during pregnancy. The changes in two endogenous markers of cell replication, proliferating cell nuclear antigen (PCNA) protein expression and bromodeoxyuridine (BrdU) incorporation, were studied. Myocyte hypertrophy was assessed by measuring the protein:DNA ratio. The expression levels of antiapoptotic regulatory proteins (BCL2 and BCL2L1) and enzymes involved in apoptosis (caspases 3, 6, 7, 9, and 10) were assessed by immunoblotting throughout gestation and postpartum. Myometrial cell apoptosis was determined by TUNEL staining and DNA fragmentation assays. Both BrdU incorporation and PCNA labeling were elevated in early pregnant myometrium and decreased dramatically after midgestation, with a simultaneous increase in cellular hypertrophy. Levels of BCL2 were high during early gestation, followed by significantly elevated levels of BCL2L1 at midgestation. The expression of caspase 10 in myometrial samples declined from a high nonpregnant level to a complete loss at early gestation. The cleaved forms of caspases (CC) 3, 6, 7, and 9, as well as poly(ADP-ribose)polymerase-1, were undetectable in the myometrial samples at early or late gestation but were transiently elevated at midgestation. Immunohistochemical staining of CC3 confirmed the activation of the caspase cascade, but TUNEL-positive staining or the increase in DNA fragmentation was not detected. Collectively, two distinct phases of myometrial growth were observed: myocyte hyperplasia associated with an increase in antiapoptotic proteins during the first half of gestation, and cellular hypertrophy during the second part of gestation. The transition between these phases was associated with transient activation of the caspase cascade that triggered the differentiation of uterine smooth muscle.     

Comparison of the accumulation of 137Cs and 90Sr by six spring wheat varieties

The uptake of ¹³⁷Cs and ⁹⁰Sr by six varieties of spring wheat (Triticum aestivum) was compared in field trials on land contaminated by the Chernobyl accident. All the experimental varieties are officially adopted for agricultural use in Belarus and are used in large-scale production. Under identical conditions of nutrition, the productivity of the varieties varied significantly by a factor of 1.3. The extent of ¹³⁷Cs and ⁹⁰Sr accumulation by wheat grain, quantified as the concentration ratio, differed between the varieties by as much as a factor of 1.6, for both radionuclides. There was a significant linear positive correlation between the ⁹⁰Sr activity concentration in grain and straw, and the calcium concentration. The correlation between ¹³⁷Cs and potassium was not significant. The results suggest that certain varieties of spring wheat used in normal agricultural practice accumulate less ¹³⁷Cs and ⁹⁰Sr into grain than others. Some spring wheat varieties accumulated relatively less ¹³⁷Cs, but did not accumulate less ⁹⁰Sr. One variety, Quattro, had a significantly lower uptake of both ⁹⁰Sr (for grain) and ¹³⁷Cs (for both grain and straw) than that of the other varieties tested. The reduction efficiency achieved by the use of these varieties, however, is not as high as that achieved by soil amelioration techniques in the past. Nevertheless, since there are no additional costs or production losses associated with these varieties, their use in the contaminated areas is worth considering as a simple, practical, and effective contribution to reducing the uptake of both ⁹⁰Sr and ¹³⁷Cs and allowing farmers to produce food-grade grain.     

Saccharomyces cerevisiae strain expressing a plant fatty acid desaturase produces polyunsaturated fatty acids and is susceptible to oxidative stress induced by lipid peroxidation

Although oxygen is essential for aerobic organisms, it also forms potentially harmful reactive oxygen species. For its simplicity, easy manipulation, and cultivation conditions, yeast is used as an attractive model in oxidative stress research. However, lack of polyunsaturated fatty acids in yeast membranes makes yeast unsuitable for research in the field of lipid peroxidation. Therefore, we have constructed a yeast strain expressing a Delta12 desaturase gene from the tropical rubber tree, Hevea brasiliensis. This yeast strain expresses the heterologous desaturase in an active form and, consequently, produces Delta9/Delta12 polyunsaturated fatty acids under inducing conditions. The functional expression of the heterologous desaturase did not affect cellular morphology or growth, indicating no general adverse effect on cellular physiology. However, the presence of polyunsaturated fatty acids changed the yeast's sensitivity to oxidative stress induced by addition of paraquat, tert-butylhydroperoxide, and hydrogen peroxide. This difference in sensitivity to the latter was followed by the formation of 4-hydroxy-2-nonenal, one of the end products of linoleic fatty acid peroxidation, which is known to play a role in cell growth control and signaling. Here we show that this yeast strain conditionally expressing the Delta12 desaturase gene provides a novel and well-defined eukaryotic model in lipid peroxidation research. Its potential to investigate the molecular basis of responses to oxidative stress, in particular the involvement of reactive aldehydes derived from fatty acid peroxidation, especially 4-hydroxy-2-nonenal, will be addressed.     

N-cadherin-based adhesion enhances Aβ release and decreases Aβ42/40 ratio

In neurons, Presenilin 1(PS1)/γ-secretase is located at the synapses, bound to N-cadherin. We have previously reported that N-cadherin-mediated cell–cell contact promotes cell-surface expression of PS1/γ-secretase. We postulated that N-cadherin-mediated trafficking of PS1 might impact synaptic PS1-amyloid precursor protein interactions and Aβ generation. In the present report, we evaluate the effect of N-cadherin-based contacts on Aβ production. We demonstrate that stable expression of N-cadherin in Chinese hamster ovary cells, expressing the Swedish mutant of human amyloid precursor protein leads to enhanced secretion of Aβ in the medium. Moreover, N-cadherin expression decreased Aβ_42/40 ratio. The effect of N-cadherin expression on Aβ production was accompanied by the enhanced accessibility of PS1/γ-secretase to amyloid precursor protein as well as a conformational change of PS1, as demonstrated by the fluorescence lifetime imaging technique. These results indicate that N-cadherin-mediated synaptic adhesion may modulate Aβ secretion as well as the Aβ_42/40 ratio via PS1/N-cadherin interactions.     

Mass spectral characterization of organophosphate-labeled lysine in peptides

Organophosphate (OP) esters bind covalently to the active site serine of enzymes in the serine hydrolase family. Recently, mass spectrometry identified covalent binding of OPs to tyrosine in a wide variety of proteins when purified proteins were incubated with OPs. In the current work, manual inspection of tandem mass spectrometry (MS/MS) data led to the realization that lysines also make a covalent bond with OPs. OP-labeled lysine residues were found in seven proteins that had been treated with either chlorpyrifos oxon (CPO) or diisopropylfluorophosphate (DFP): human serum albumin (K212, K414, K199, and K351), human keratin 1 (K211 and K355), human keratin 10 (K163), bovine tubulin alpha (K60, K336, K163, K394, and K401), bovine tubulin beta (K58), bovine actin (K113, K291, K326, K315, and K328), and mouse transferrin (K296 and K626). These results suggest that OP binding to lysine is a general phenomenon. Characteristic fragments specific for CPO-labeled lysine appeared at 237.1, 220.0, 192.0, 163.9, 128.9, and 83.9 amu. Characteristic fragments specific for DFP-labeled lysine appeared at 164.0, 181.2, and 83.8 amu. This new OP-binding motif to lysine suggests new directions to search for mechanisms of long-term effects of OP exposure and in the search for biomarkers of OP exposure.     

Allosteric Modulation of PS1/γ-Secretase Conformation Correlates with Amyloid β42/40 Ratio

Background

Presenilin 1(PS1) is the catalytic subunit of γ-secretase, the enzyme responsible for the Aβ C-terminal cleavage site, which results in the production of Aβ peptides of various lengths. Production of longer forms of the Aβ peptide occur in patients with autosomal dominant Alzheimer disease (AD) due to mutations in presenilin. Many modulators of γ-secretase function have been described. We hypothesize that these modulators act by a common mechanism by allosterically modifying the structure of presenilin.

Methodology/Principal Findings

To test this hypothesis we generated a genetically encoded GFP-PS1-RFP (G-PS1-R) FRET probe that allows monitoring of the conformation of the PS1 molecule in its native environment in live cells. We show that G-PS1-R can be incorporated into the γ-secretase complex, reconstituting its activity in PS1/2 deficient cells. Using Förster resonance energy transfer (FRET)-based approaches we show that various pharmacological and genetic manipulations that target either γ-secretase components (PS1, Pen2, Aph1) or γ-secretase substrate (amyloid precursor protein, APP) and are known to change Aβ₄₂ production are associated with a consistent conformational change in PS1.

Conclusions/Significance

These results strongly support the hypothesis that allosteric changes in PS1 conformation underlie changes in the Aβ_42/40 ratio. Direct measurement of physiological and pathological changes in the conformation of PS1/γ-secretase may provide insight into molecular mechanism of Aβ₄₂ generation, which could be exploited therapeutically.     

Comparison of 61 Sequenced Escherichia coli Genomes

Escherichia coli is an important component of the biosphere and is an ideal model for studies of processes involved in bacterial genome evolution. Sixty-one publically available E. coli and Shigella spp. sequenced genomes are compared, using basic methods to produce phylogenetic and proteomics trees, and to identify the pan- and core genomes of this set of sequenced strains. A hierarchical clustering of variable genes allowed clear separation of the strains into clusters, including known pathotypes; clinically relevant serotypes can also be resolved in this way. In contrast, when in silico MLST was performed, many of the various strains appear jumbled and less well resolved. The predicted pan-genome comprises 15,741 gene families, and only 993 (6%) of the families are represented in every genome, comprising the core genome. The variable or ‘accessory’ genes thus make up more than 90% of the pan-genome and about 80% of a typical genome; some of these variable genes tend to be co-localized on genomic islands. The diversity within the species E. coli, and the overlap in gene content between this and related species, suggests a continuum rather than sharp species borders in this group of Enterobacteriaceae.     

Electroporation-Induced Inward Current in Voltage-Clamped Guinea Pig Ventricular Myocytes

Electroporation induced by high-strength electrical fields has long been used to investigate membrane properties and facilitate transmembrane delivery of molecules and genes for research and clinical purposes. In the heart, electric field-induced passage of ions through electropores is a factor in defibrillation and postshock dysfunction. Voltage-clamp pulses can also induce electroporation, as exemplified by findings in earlier studies on rabbit ventricular myocytes: Long hyperpolarizations to ≤-110?mV induced influx of marker ethidium and irregular inward currents that were as large with external NMDG(+) as Na(+). In the present study, guinea pig ventricular myocytes were bathed with NMDG(+), Na(+) or NMDG(+)?+?La(3+) solution (36°C) and treated with five channel blockers. Hyperpolarization of myocytes in NMDG(+) solution elicited an irregular inward current (I (ep)) that reversed at -21.5?±?1.5?mV. In myocytes hyperpolarized with 200-ms steps every 30?s, I (ep) occurred in "episodes" that lasted for one to four steps. Boltzmann fits to data on the incidence of I (ep) per experiment indicate 50% incidence at -129.7?±?1.4?mV (Na(+)) and -146.3?±?1.6?mV (NMDG(+)) (slopes ≈-7.5?mV). I (ep) amplitude increased with negative voltage and was larger with Na(+) than NMDG(+) (e.g., -2.83?±?0.34 vs. -1.40?±?0.22?nA at -190?mV). La(3+) (0.2?mM) shortened episodes, shifted 50% incidence by -35?mV and decreased amplitude, suggesting that it inhibits opening/promotes closing of electropores. We compare our findings with earlier ones, especially in regard to electropore selectivity. In the Appendix, relative permeabilities and modified excluded-area theory are used to derive estimates of electropore diameters consistent with reversal potential -21.5?mV.     

Dimethylaminopyridine derivatives of lupane triterpenoids are potent disruptors of mitochondrial structure and function

Development of mitochondrially-targeted drugs is receiving increasing attention because of the central roles these organelles play in energy production, reactive oxygen generation, and regulation of cell death pathways. Previous studies have demonstrated that both natural and synthetic triterpenoids can disrupt mitochondrial structure and function. In this study, we tested the ability of a number of dimethylaminopyridine (DMAP) derivatives of lupane triterpenoids to target mitochochondria in two human melanoma cell lines and an untransformed normal fibroblast line. These compounds induced a striking fragmentation and depolarization of the mitochondrial network, along with an inhibition of cell proliferation. A range of potencies among these compounds was noted, which was correlated with the number, position, and orientation of the DMAP groups. Overall, the extent of proliferation inhibition mirrored the effectiveness of mitochondrial disruption. Thus, DMAP derivatives of lupane triterpenoids can be potent mitochondrial perturbants that appear to suppress cell growth primarily via their mitochondrial effects.