3H]L-657,743 (MK-912): a new, high affinity, selective radioligand for brain alpha 2-adrenoceptors

L-657,743 (MK-912), a highly potent and selective alpha 2-adrenoceptor antagonist was tritiated to a high specific activity and its binding characteristics to brain tissue were determined. The specific binding of [3H]L-657,743 to rat cerebrocortex was saturable, reversible, and dependent on tissue concentration. In saturation studies, [3H]L-657,743 binding was resolved into two high affinity components exhibiting Kd values of 86 pM and 830 pM with densities of 82 fmol/mg protein and 660 fmol/mg protein, respectively. Based on the binding potencies of a variety of compounds with differing receptor selectivities, the sites labeled by [3H]L-657,743 were characteristic of alpha 2-adrenoceptors. In contrast to alpha 2-antagonists, alpha 2-agonists displayed shallow competition curves. In the presence of 100 microM GTP, Gpp(NH)p or 150 mM NaCl, the competition curve for epinephrine was shifted to the right, whereas that for yohimbine was unaffected. In studies utilizing human cerebrocortical tissue, [3H]L-657,743 also bound with high affinity to sites characteristic of alpha 2-adrenoceptors.     

A highly conserved nuclear gene for low-level phylogenetics: elongation factor-1 alpha recovers morphology-based tree for heliothine moths

Molecular systematists need increased access to nuclear genes. Highly conserved, low copy number protein-encoding nuclear genes have attractive features for phylogenetic inference but have heretofore been applied mostly to very ancient divergences. By virtue of their synonymous substitutions, such genes should contain a wealth of information about lower-level taxonomic relationships as well, with the advantage that amino acid conservatism makes both alignment and primer definition straightforward. We tested this postulate for the elongation factor-1 alpha (EF-1 alpha) gene in the noctuid moth subfamily Heliothinae, which has probably diversified since the middle Tertiary. We sequenced 1,240 bp in 18 taxa representing heliothine groupings strongly supported by previous morphological and allozyme studies. The single most parsimonious gene tree and the neighbor-joining tree for all nucleotides show almost complete concordance with the morphological tree. Homoplasy and pairwise divergence levels are low, transition/transversion ratios are high, and phylogenetic information is spread evenly across gene regions. The EF-1 alpha gene and presumably other highly conserved genes hold much promise for phylogenetics of Tertiary age eukaryote groups.      

A protonmotive force as the source of energy for galactoside transport in energy depletedEscherichia Coli

Summary Uracil transport inSaccharomyces cerevisiae is mediated by a specific permease which does not recognize other pyrimidines such as uridine, cytosine, thymine, 2-hydroxypyrimidine or 5-amino-uracil; hypoxanthine and 6-amino-uracil slightly inhibit the uptake of uracil in a strain lacking cytosine permease activity. Wild type cells concentrate extracellular uracil before its transformation into UMP and subsequent incorporation into nucleic acids. A strain lacking UMP pyrophosphorylase and uridine ribohydrolase (strainfur 1–8 rh, in which the endogenous production as well as the utilization of uracil are lacking) is able to concentrate¹⁴C-2 uracil from the medium. At the same time no other¹⁴C-2 labelled compound could be detected in this strain, thus suggesting that the uptake of uracil in yeast occurs by active transport which is not coupled to the UMP pyrophosphorylase. The optimal pH of uracil uptake in standard growth conditions was 4.3. It was deduced from experiments performed on strainfur 1–8 rh with³H-5 and¹⁴C-2 uracil that the intracellular pool of uracil is recycled once the steady-state has been reached. First order kinetics with similar rate constants were observed for uracil efflux in strainfur 1–8 rh (k min^–1=0.75±0.08) as well as in the strain lacking uracil permease,fur 1–8 rh fur 4–6 (k min^–1=0.60±0.08). The intracellular pool of¹⁴C-2 uracil can be chased in strainfur 1–8 rh by addition of³H uracil without inducing a large initial acceleration of the exit rate (the rate constant remained at 0.60). 2-4-dinitrophenol inhibits the uptake of uracil but also reduces the efflux of uracil in strainfur 1–8 rh fur 4–6. These data and the comparison with cytosine transport in the same organism support the hypothesis that, whereas uracil uptake is a permease mediated active transport, the efflux of uracil does not involve the uracil uptake permease. A coefficient of permeability of 7.4×10^–7 cm sec^–1 was calculated for uracil.     

Plant virus infection development as affected by heavy metal stress

The work was focused on the investigation of possible dependencies between the development of viral infection in plants and the presence of high heavy metal concentrations in soil. Field experiments have been conducted in order to study the development of systemic tobacco mosaic virus (TMV) infection in Lycopersicon esculentum L. cv. Miliana plants under effect of separate salts of heavy metals Cu, Zn and Pb deposited in soil. As it is shown, simultaneous effect of viral infection and heavy metals in tenfold maximum permissible concentration leads to decrease of total chlorophyll content in experiment plants mainly due to the degradation of chlorophyll a. The reduction of chlorophyll concentration under the combined influence of both stress factors was more serious comparing to the separate effect of every single factor. Plants' treatment with toxic concentrations of lead and zinc leaded to slight delay in the development of systemic TMV infection together with more than twofold increase of virus content in plants that may be an evidence of synergism between these heavy metal's and virus' effects. Contrary, copper although decreased total chlorophyll content but showed protective properties and significantly reduced amount of virus in plants.     

Tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone promotes functional cooperation of Bcl2 and c-Myc through phosphorylation in regulating cell survival and proliferation

Nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is formed by nitrosation of nicotine and has been identified as the most potent carcinogen contained in cigarette smoke. NNK significantly contributes to smoking-related lung cancer, but the molecular mechanism remains enigmatic. Bcl2 and c-Myc are two major oncogenic proteins that cooperatively promote tumor development. We report here that NNK simultaneously stimulates Bcl2 phosphorylation exclusively at Ser(70) and c-Myc at Thr(58) and Ser(62) through activation of both ERK1/2 and PKCalpha, which is required for NNK-induced survival and proliferation of human lung cancer cells. Treatment of cells with staurosporine or PD98059 blocks both Bcl2 and c-Myc phosphorylation and results in suppression of NNK-induced proliferation. Specific depletion of c-Myc expression by RNA interference retards G(1)/S cell cycle transition and blocks NNK-induced cell proliferation. Phosphorylation of Bcl2 at Ser(70) promotes a direct interaction between Bcl2 and c-Myc in the nucleus and on the outer mitochondrial membrane that significantly enhances the half-life of the c-Myc protein. Thus, NNK-induced functional cooperation of Bcl2 and c-Myc in promoting cell survival and proliferation may occur in a novel mechanism involving their phosphorylation, which may lead to development of human lung cancer and/or chemoresistance.     

Promoter DNA Methylation Regulates Murine SUR1 (Abcc8) and SUR2 (Abcc9) Expression in HL-1 Cardiomyocytes

Two mammalian genes encode the SURx (SUR1, Abcc8 and SUR2, Abcc9) subunits that combine with Kir6.2 (Kcnj11) subunits to form the ATP-sensitive potassium (KATP) channel in cardiac myocytes. Different isoform combinations endow the channel with distinct physiological and pharmacological properties, and we have recently reported that the molecular composition of sarcolemmal KATP channels is chamber specific in the mouse heart. KATP channel composition is determined by what subunits are expressed in a cell or tissue. In the present study, we explore the role of CpG methylation in regulating SUR1 and SUR2 expression. In HL-1 cardiomyocytes, as in atrial myocytes, SUR1 expression is markedly greater than SUR2. Consistent with CpG methylation-dependent silencing of SUR2 expression, bisulfite sequencing of genomic DNA isolated from HL-1 cells demonstrates that 57.6% of the CpGs in the promoter region of the SUR2 gene are methylated, compared with 0.14% of the the CpG residues in the SUR1 sequence. Moreover, treatment with 10 μM 5-aza-2'-deoxycytidine (Aza-dC) significantly increased both the unmethylated fraction of the SUR2 CpG island and mRNA expression. However, we cannot rule out additional mechanisms of Aza-dC action, as Aza-dC also causes a decrease in SUR1 expression and lower doses of Aza-dC do not alter the unmethylated DNA fraction but do elicit a small increase in SUR2 expression. The conclusion that DNA methylation alone is not the only regulator of SUR subunit expression is also consistent with observations in native myocytes, where the CpG islands of both SUR genes are essentially unmethylated in both atrial and ventricular myocytes. Collectively, these data demonstrate the potential for CpG methylation to regulate SURx subunit expression and raises the possibility that regulated or aberrant CpG methylation might play a role in controlling channel structure and function under different physiological conditions or different species.