Re-expression of alpha skeletal actin as a marker for dedifferentiation in cardiac pathologies

Differentiation of foetal cardiomyocytes is accompanied by sequential actin isoform expression, i.e. down-regulation of the 'embryonic' alpha smooth muscle actin, followed by an up-regulation of alpha skeletal actin (αSKA) and a final predominant expression of alpha cardiac actin (αCA). Our objective was to detect whether re-expression of αSKA occurred during cardiomyocyte dedifferentiation, a phenomenon that has been observed in different pathologies characterized by myocardial dysfunction. Immunohistochemistry of αCA, αSKA and cardiotin was performed on left ventricle biopsies from human patients after coronary bypass surgery. Furthermore, actin isoform expression was investigated in left ventricle samples of rabbit hearts suffering from pressure- and volume-overload and in adult rabbit ventricular cardiomyocytes during dedifferentiation in vitro . Atrial goat samples up to 16 weeks of sustained atrial fibrillation (AF) were studied ultrastructurally and were immunostained for αCA and αSKA. Up-regulation of αSKA was observed in human ventricular cardiomyocytes showing down-regulation of αCA and cardiotin. A patchy re-expression pattern of αSKA was observed in rabbit left ventricular tissue subjected to pressure- and volume-overload. Dedifferentiating cardiomyocytes in vitro revealed a degradation of the contractile apparatus and local re-expression of αSKA. Comparable αSKA staining patterns were found in several areas of atrial goat tissue during 16 weeks of AF together with a progressive glycogen accumulation at the same time intervals. The expression of αSKA in adult dedifferentiating cardiomyocytes, in combination with PAS-positive glycogen and decreased cardiotin expression, offers an additional tool in the evaluation of myocardial dysfunction and indicates major changes in the contractile properties of these cells.     

Genetic variation in postfire aspen seedlings in yellowstone national park

A rare episode of regeneration of aspen (Populus tremuloides Michx.) by seeds occurred in Yellowstone National Park (YNP), Wyoming, USA, following extensive fires that occurred in 1988. In 1997, we sampled 410 aspen seedlings from 23 local populations distributed widely across YNP to determine how genetic diversity varies with elevation, substrate, plant competition, ungulate browsing, and geographical location. We employed 132 randomly amplified polymorphic DNA (RAPD) markers based on six primers to show genetic relationships within and among the postfire aspen seedling populations. Measures of genetic variation, including estimates of percentage polymorphic loci, expected heterozygosity, and Nei's FST, indicated that most of the variation occurred within rather than among local populations. There was no indication of geographical differentiation among sampled populations based on hierarchal estimates of Nei's FST, neighbour-joining, or correlations between genetic distance and geographical distance. Even genetically distant populations shared nearly 90% of the same markers. Within plots, the amount of genetic variation decreased slightly in response to increased percentage vegetative cover, mean seedling basal diameter, and mean seedling height. Geological substrate, density of lodgepole pine (Pinus contorta var. latifolia Dougl.) seedlings, browsing intensity, and elevation were not significantly related to levels of genetic variation within the seedling plots. These data suggest that genetic variation and geographical structure among seedling populations may occur over time as the transition from seedling-dominated stands to clone-dominated stands occurs.     

Phytochemistry of the mopane,Colophospermum mopane

The polyphenolic pool of the heartwood of the mopane, Colophospermum mopane Kirk ex J. Leonard, exhibits extreme diversity and complexity. It comprises a variety of monomeric flavonoids, e.g. flavan-3-ols, flavan-3,4-diols including the mopanols and peltogynols, flavonols, dimeric proanthocyanidins, e.g. proguibourtinidins, profisetinidins, promopanidins, propeltogynidins, and a variety of profisetinidin-type triflavanoids. The di- and tri-meric proanthocyanidins are accompanied by several functionalized tetrahydropyrano- and hexahydrodipyrano-chromenes (phlobatannins) that originate from the bi- and tri-flavanoids, respectively, via rearrangement of the pyran heterocycle(s). Owing to the predominance of the 5-deoxy (A-ring) flavan-3-ols, the chain terminating moieties in the biosynthesis of oligo- and poly-meric proanthocyanidins, the di- and tri-meric analogs also exhibit diversity as far as interflavanyl bonding positions are concerned. Such heterogeneity results from the reduced nucleophilicity of the A-rings of 5-deoxy flavan-3-ols, compared to the A-rings of the 5-oxy analogs (catechins), hence permitting alternative centers to participate in proanthocyanidin formation. Biomimetic-type syntheses were extensively utilized to unequivocally establish constitution and absolute stereochemistry of both the conventional and pyran ring rearranged-type di- and tri-meric compounds. Comprehension of the intricate mechanistic and stereochemical course of the pyran ring rearrangement reactions also contributed significantly to unambiguous structure elucidations. The aerial parts of the mopane are rich in essential oils that comprise mainly alpha-pinene and limonene, which are presumably responsible for the strong turpentine odor of the pods. The leaves also contain significant concentrations of beta-sitosterol and stigmasterol which are apparently the source of sterols in various organs of the mopane moth, Gonimbrasia belina. Three diterpenes, dihydrogrindelic acid, labd-13E-en-15-oate and dihydrogrindelaldehyde are present in the bark and seeds, the latter compound exhibiting significant cytotoxicity against a human breast cancer cell line.     

Bcl-2 family member Bfl-1/A1 sequesters truncated bid to inhibit is collaboration with pro-apoptotic Bak or Bax

Following caspase-8 mediated cleavage, a carboxyl-terminal fragment of the BH3 domain-only Bcl-2 family member Bid transmits the apoptotic signal from death receptors to mitochondria. In a screen for possible regulators of Bid, we defined Bfl-1/A1 as a potent Bid interacting protein. Bfl-1 is an anti-apoptotic Bcl-2 family member, whose preferential expression in hematopoietic cells and endothelium is controlled by inflammatory stimuli. Its mechanism of action is unknown. We find that Bfl-1 associates with both full-length Bid and truncated (t)Bid, via the Bid BH3 domain. Cellular expression of Bfl-1 confers protection against CD95- and Trail receptor-induced cytochrome c release. In vitro assays, using purified mitochondria and recombinant proteins, demonstrate that Bfl-1 binds full-length Bid, but does not interfere with its processing by caspase-8, or with its mitochondrial association. Confocal microscopy supports that Bfl-1, which at least in part constitutively localizes to mitochondria, does not impede tBid translocation. However, Bfl-1 remains tightly and selectively bound to tBid and blocks collaboration between tBid and Bax or Bak in the plane of the mitochondrial membrane, thereby preventing mitochondrial apoptotic activation. Lack of demonstrable interaction between Bfl-1 and Bak or Bax in the mitochondrial membrane suggests that Bfl-1 generally prevents the formation of a pro-apoptotic complex by sequestering BH3 domain-only proteins.     

Serum lactate dehydrogenase isoenzyme 1 activity in patients with testicular germ cell tumors correlates with the total number of copies of the short arm of chromosome 12 in the tumor

Summary The aim of our study was to assess the relationship between the serum lactate dehydrogenase isoenzyme 1 (S-LDH-1) activity in patients with testicular germ cell tumors and the number of copies of the short arm of chromosome 12 (12p) present in tumor. Twenty-seven adult patients with measurable tumor lesions were studied. Twenty-five had three or more copies of chromosome 12 per cell in the tumors. Nineteen had one or more copies of a specific chromosomal abnormality, an isochromosome of the short arm of chromosome 12, i(12p). Fourteen had increased S-LDH-1 levels. S-LDH-1 activity correlated significantly with the product of total tumor volume and the total number of copies of the short arm of chromosome 12 present per cell (total tumor 12p). We conclude that the total number of copies of the short arm of chromosome 12 in the tumors is most probably a factor contributing to the LDH-1 activity released from the tumors.     

Synthetic, electrochemical and structural aspects of a series of ferrocene-containing dicarbonyl β-diketonato rhodium(I) complexes

Treatment of [Rh(β-diketonato)(cod)] with CO resulted in better yields of [Rh(FcCOCHCOR)(CO)₂] than by treating [Rh(Cl)(CO)₂]₂ with FcCOCH₂COR, R = CF₃ (Hfctfa), CH₃ (Hfca), Ph (Hbfcm, Ph = phenyl) and Fc (Hdfcm, Fc = ferrocenyl). The single crystal structure of the fctfa rhodium(I) complex [C₁₆H₁₀F₃FeO₄Rh], monoclinic, C 2/c(15), a = 13.266(3) Å, b = 19.553(3) Å, c = 13.278(3) Å, β = 100.92(2)°, Z = 8 showed both rotational and translational displacement disorders for the CF₃ group. An electrochemical study revealed that the formal reduction potential, E⁰′, for the electrochemically reversible one electron oxidation of the ferrocenyl group varied between 0.304 (for the fctfa complex) and 0.172 V (for the dfcm complex) versus Fc/Fc⁺ in a manner that could be directly traced to the group electronegativities, χ_R, of the R groups on the β-diketonato ligands, as well as to the values of the free β-diketones. Anodic peak potentials, E_pa,Rh, for the dominant cyclic voltammetry peak associated with rhodium(I) oxidation were between 0.718 (bfcm complex) and 1.022 V (dfcm complex) versus Fc/Fc⁺. Coulometric experiments implicated a second, much less pronounced anodic wave for the apparent two-electron Rh^I oxidation that overlaps with the ferrocenyl anodic wave and that the redox processes associated with these two Rh^I oxidation waves are in slow equilibrium with each other.     

Circular dichroism, a powerful tool for the assessment of absolute configuration of flavonoids

Circular dichroism is a powerful tool for establishing the absolute configuration of flavonoids and proanthocyanidin analogues. It has been utilized to study the configuration of flavanones, dihydroflavonols (3-hydroxyflavanones), flavan-3-ols, flavan-4-ols, flavan-3,4-diols, flavans, isoflavans, isoflavanones, pterocarpans, 6a-hydroxypterocarpans, rotenoids, 12a-hydroxyrotenoids, neoflavonoids, 3,4-dihydro-4-arylcoumarins, 4-arylflavan-3-ols, auronols, homoisoflavanones, proanthocyanidins, and various classes of biflavonoids. Results relevant to the correlation of circular dichroic data and the absolute configuration of the diastereoisomers of some of the above classes of compounds will be discussed.     

Heterogeneity of the interflavanyl bond in proanthocyanidins from natural sources lacking C-4 (C-ring) deoxy flavonoid nucleophiles

The proanthocyanidin pool in the floral kingdom usually involves the presence of carbon-carbon bonds linking predominantly flavan-3-ol constituent moieties. Such an ensemble of flavan-3-ol units originates via electrophilic aromatic substitution of flavan-4-yl carbocations (or their equivalents) derived from flavan-4-ols and/or flavan-3,4-diols and the nucleophilic centers of the m-oxygenated A-rings of flavan-3-ol nucleophiles. In the absence of these potent flavan-3-ol nucleophiles with their aptitude for the formation of carbon-carbon bonds, alternative centers emerge as participants in interflavanyl bond formation. Such a phenomenon is demonstrated for the distribution of various profisetinidin-, prorobinetinidin-, proguibourtinidin-, promelacacinidin- and proteracacinidin-type pro- and leuco-anthocyanidins in several southern hemisphere heartwood species.     

Landscape Patterns of Sapling Density,Leaf Area,and Aboveground Net Primary Production in Postfire Lodgepole Pine Forests,Yellowstone National Park (USA)

Causes and implications of spatial variability in postfire tree density and understory plant cover for patterns of aboveground net primary production (ANPP) and leaf area index (LAI) were examined in ninety 11-year-old lodgepole pine (Pinus contorta var. latifolia Engelm.) stands across the landscape of Yellowstone National Park (YNP), Wyoming, USA. Field studies and aerial photography were used to address three questions: (1) What is the range and spatial pattern of lodgepole pine sapling density across the burned Yellowstone landscape and what factors best explain this variability? (2) How do ANPP and LAI vary across the landscape and is their variation explained by abiotic factors, sapling density, or both? (3) What is the predicted spatial pattern of ANPP and LAI across the burned Yellowstone landscape? Stand density spanned six orders of magnitude, ranging from zero to 535,000 saplings ha^?1, and it decreased with increasing elevation and with increasing distance from unburned forest (r ²?=?0.37). Postfire densities mapped from 1:30,000 aerial photography revealed that 66% of the burned area had densities less than 5000 saplings ha^?1 and approximately 25% had densities greater than 10,000 saplings ha^?1; stand density varied spatially in a fine-grained mosaic. New allometric equations were developed to predict aboveground biomass, ANPP, and LAI of lodgepole pine saplings and the 25 most common herbaceous and shrub species in the burned forests. These allometrics were then used with field data on sapling size, sapling density, and percent cover of graminoid, forb, and shrub species to compute stand-level ANPP and LAI. Total ANPP averaged 2.8 Mg ha^?1y^?1 but ranged from 0.04 to 15.12 Mg ha^?1y^?1. Total LAI averaged 0.80 m² m^?2 and ranged from 0.01 to 6.87 m² m^?2. Variation in ANPP and LAI was explained by both sapling density and abiotic factors (elevation and soil class) (ANOVA, r ²?=?0.80); abiotic variables explained 51%–54% of this variation. The proportion of total ANPP contributed by herbaceous plants and shrubs declined sharply with increasing sapling density (r ²?=?0.72) and increased with elevation (r ²?=?0.36). However, total herbaceous productivity was always less than 2.7 Mg ha^?1 y^?1, and herbaceous productivity did not compensate for tree production when trees were sparse. When extrapolated to the landscape, 68% of the burned landscape was characterized by ANPP values less than 2.0 Mg ha^?1y^?1, 22% by values ranging from 2 to 4 Mg ha^?1y^?1, and the remaining 10% by values greater than 4 Mg ha^?1y^?1. The spatial patterns of ANPP and LAI were less heterogeneous than patterns of sapling density but still showed fine-grained variation in rates. For some ecosystem processes, postfire spatial heterogeneity within a successional stage may be similar in magnitude to the temporal variation observed through succession.     

1H/31P Polarization Transfer at 9.4 Tesla for Improved Specificity of Detecting Phosphomonoesters and Phosphodiesters in Breast Tumor Models

Purpose

To assess the ability of a polarization transfer (PT) magnetic resonance spectroscopy (MRS) technique to improve the detection of the individual phospholipid metabolites phosphocholine (PC), phosphoethanolamine (PE), glycerophosphocholine (GPC), and glycerophosphoethanolamine (GPE) in vivo in breast tumor xenografts.

Materials and Methods

The adiabatic version of refocused insensitive nuclei enhanced by polarization transfer (BINEPT) MRS was tested at 9.4 Tesla in phantoms and animal models. BINEPT and pulse-acquire (PA) ³¹P MRS was acquired consecutively from the same orthotopic MCF-7 (n = 10) and MDA-MB-231 (n = 10) breast tumor xenografts. After in vivo MRS measurements, animals were euthanized, tumors were extracted and high resolution (HR)-MRS was performed. Signal to noise ratios (SNRs) and metabolite ratios were compared for BINEPT and PA MRS, and were also measured and compared with that from HR-MRS.

Results

BINEPT exclusively detected metabolites with ¹H-³¹P coupling such as PC, PE, GPC, and GPE, thereby creating a significantly improved, flat baseline because overlapping resonances from immobile and partly mobile phospholipids were removed without loss of sensitivity. GPE and GPC were more accurately detected by BINEPT in vivo, which enabled a reliable quantification of metabolite ratios such as PE/GPE and PC/GPC, which are important markers of tumor aggressiveness and treatment response.

Conclusion

BINEPT is advantageous over PA for detecting and quantifying the individual phospholipid metabolites PC, PE, GPC, and GPE in vivo at high magnetic field strength. As BINEPT can be used clinically, alterations in these phospholipid metabolites can be assessed in vivo for cancer diagnosis and treatment monitoring.