Identification and characterization of (3H)-rauwolscine binding to alpha 2-adrenoceptors in the canine saphenous vein

The biochemical exploration of the alpha 2-adrenergic receptors was investigated in the canine saphenous vein using the highly selective alpha 2-adrenergic antagonist rauwolscine as a tritiated ligand. Following an enzymatic digestive pretreatment, we isolated a purified smooth muscle cell membrane fraction from saphenous veins in quantity sufficient to permit us to study the venous alpha 2-adrenoceptor content. The binding of tritiated rauwolscine was rapid, specific, saturable and reversible. The presence of high affinity binding sites (Kd = 1.53 +/- 0.71 nM) with a density of binding Bmax of 125.2 +/- 43.1 fmol/mg protein was demonstrated on a unique class of non interacting sites (nHill = 1.001 +/- 0.06). The kinetically derived Kd was 1.28 nM, in good agreement with the value obtained from saturation isotherms. The pharmacological profile of these sites was assessed by the comparison of the potency of alpha-adrenergic agonists and antagonists to inhibit 1 nM (3H)-rauwolscine. Their efficacy was respectively: rauwolscine greater than phentolamine greater than RX 781094 greater than clonidine much greater than prazosin greater than (-)-phenylephrine greater than (-)-noradrenaline. The results showed that (3H)-rauwolscine bound specifically to sites in our membranal preparation, which had the pharmacological characteristics of the alpha 2-adrenoceptors. The correlation between biochemical and pharmacological data revealed the usefulness of binding methods in the further study of adrenergic mechanisms in the canine saphenous vein.     

Interaction of the p85 subunit of PI 3-kinase and its N-terminal SH2 domain with a PDGF receptor phosphorylation site: structural features and analysis of conformational changes.

Circular dichroism and fluorescence spectroscopy were used to investigate the structure of the p85 alpha subunit of the PI 3-kinase, a closely related p85 beta protein, and a recombinant SH2 domain-containing fragment of p85 alpha. Significant spectral changes, indicative of a conformational change, were observed on formation of a complex with a 17 residue peptide containing a phosphorylated tyrosine residue. The sequence of this peptide is identical to the sequence surrounding Tyr751 in the kinase-insert region of the platelet-derived growth factor beta-receptor (beta PDGFR). The rotational correlation times measured by fluorescence anisotropy decay indicated that phosphopeptide binding changed the shape of the SH2 domain-containing fragment. The CD and fluorescence spectroscopy data support the secondary structure prediction based on sequence analysis and provide evidence for flexible linker regions between the various domains of the p85 proteins. The significance of these results for SH2 domain-containing proteins is discussed.     

Effects of acronycine on nucleic acid synthesis and population growth in mammalian tumor cell cultures

Acronycine — an alkaloid with antineoplastic activity against a wide range of experimental tumors — at concentrations of 0.5-12 μg/ml rapidly inhibits RNA synthesis in L5178Y mouse lymphoma and IRC rat monocytic leukemia cultures. Culture growth is arrested only at acronycine concentrations which markedly inhibit RNA synthesis. DNA synthesis is inhibited at rather higher concentrations but this is not a prerequisite of the arrest of growth. It is suggested that the arrest of growth may be a consequence of the inhibition of RNA synthesis. In both cultures arrest of growth coincides with the appearance of many cells with two apparently normal nuclei. Cells are not arrested in mitosis. It is shown these binucleated cells very probably arise from an inhibition of cell cleavage. Studies with synchronized cultures show that at low drug concentrations, more than one cell cycle may elapse before growth is arrested and binucleated cells appear, indicating the effect on cytokinesis is not immediate. The results suggest that the arrest of growth may be a result of a slow depletion of a component essential for cell cleavage. The disturbance at division is a major factor in arresting growth at low drug concentrations. At higher acronycine concentrations, when RNA synthesis may be inhibited by 80–90%, the cytotoxic effects appear earlier and are less specifically directed at cytokinesis; DNA synthesis is then also rapidly and markedly inhibited.     

Bioactive recombinant methionyl bovine prolactin: structure-function studies using site-specific mutagenesis

A method has been developed for the extraction from transformed Escherichia coli cells of methionyl bovine PRL (met-bPRL) in a relatively pure form. While the extracted met-bPRL was as reactive as the native hormone with respect to polyclonal anti-bPRL antibodies, its bioactivity, as measured by the Nb2 lactogen in vitro bioassay, was relatively low. The bioactivity of the met-bPRL could be increased to the same order as that of the native hormone by treatment with a mixture of oxidized and reduced thioredoxin. A number of variant met-bPRLs containing specific amino acid changes have been generated by site-specific mutagenesis. The changes involved the substitution (or deletion) of some of the conserved amino acids in bPRL by the different amino acids present at the corresponding positions in the related, but nonlactogenic bovine GH. Nine mutants containing single amino acid changes had bio- and immunoactivities of the same order as those of met-bPRL. One mutant, which incorporated two of the single amino acid changes (serine 62 to threonine and threonine 65 to alanine), had immunoactivity approximating that of met-bPRL but much lower bioactivity (45%). A further mutant, generated by the deletion of tyrosine 28, had essentially no bioactivity although it could not be distinguished immunologically from met-bPRL or bPRL. The findings are discussed in the light of the putative three-dimensional PRL structure and current hypotheses which seek to relate specific regions of PRL to lactogenic activity. It appears that the first putative alpha-helix of bPRL is important for the binding and mitogenic activity of the hormone.     

PI 3-kinase is a dual specificity enzyme: autoregulation by an intrinsic protein-serine kinase activity.

Phosphatidylinositol 3-kinase (PI 3-kinase) has a regulatory 85 kDa adaptor subunit whose SH2 domains bind phosphotyrosine in specific recognition motifs, and a catalytic 110 kDa subunit. Mutagenesis of the p110 subunit, within a sequence motif common to both protein and lipid kinases, demonstrates a novel intrinsic protein kinase activity which phosphorylates the p85 subunit on serine at a stoichiometry of approximately 1 mol of phosphate per mol of p85. This protein-serine kinase activity is detectable only upon high affinity binding of the p110 subunit with its unique substrate, the p85 subunit. Tryptic phosphopeptide mapping revealed that the same major peptide was phosphorylated in p85 alpha both in vivo in cultured cells and in the purified recombinant enzyme. N-terminal sequence and mass analyses were used to identify Ser608 as the major phosphorylation site on p85 alpha. Phosphorylation of the p85 subunit at this serine causes an 80% decrease in PI 3-kinase activity, which can subsequently be reversed upon treatment with protein phosphatase 2A. These results have implications for the role of inter-subunit serine phosphorylation in the regulation of the PI 3-kinase in vivo.     

Osmotic adjustment in Fraxinus excelsior L . : malate and mannitol accumulation in leaves under drought conditions

 In leaves of Fraxinus excelsior L., malate and mannitol were characterized by ¹³C NMR spectroscopy and enzymatic specific assays as the major constituents of a soluble carbon fraction involved in an osmotic adjustment. During a summer drought where predawn leaf water potential of adult trees growing in a mesoxerophilic stand fell to – 4 MPa in August, malate and mannitol leaf contents increased by a factor of 1.8 and 2.2 respectively, compared to control trees growing on a flood plain. This drought stress led to concentrations as high as 280 mM and 600 mM for mannitol and malate, respectively. The effects of gradually developing water deficit were also studied in a semi-controlled environment in 3-year-old seedlings. When predawn leaf water potential reached -6 MPa, leaves displayed a low turgor pressure but stomatal conductance was still measurable. Malate and mannitol were also the main osmoticum involved. After rewatering, gas exchange capacities were largely restored. Altogether, these results show that the strong water-stress tolerance of Fraxinus excelsior is in part related to an accumulation of malate and mannitol. Received: 3 January 1996 / Accepted: 19 March 1996     

Glycosylated equine prolactin and its carbohydrate moiety

Glycosylated equine prolactin (G-ePRL) and nonglycosylated ePRL were purified to homogeneity from side fractions obtained during isolation of LH/FSH from horse pituitaries. Both PRL forms were isolated together in high yield by the isolation procedure used for glycosylated porcine PRL/(G-pPRL) and pPRL, involving acetone extraction/precipitation, NaCl and isoelectric precipitation, and gel filtration. Purification of G-ePRL required additional Con A chromatography. The N-terminal amino acid sequencing for 32 cycles of G-ePRL and ePRL resulted in sequences identical to the known primary structure of ePRL. Based on MALDI mass spectrometry analysis and SDS-PAGE mobilities,G-ePRL and ePRL had estimated molecular weights of 25,000 and 23,000 Da, respectively. G-ePRL displayed only 60% of the immunoreactivity of ePRL in homologous radioimmunoassay. Using the Nb2 lymphoma cell bioassay, ePRL was found to have about l/30th the mitogenic activity of bovine PRL; G-ePRL was approximately l/10th as active as ePRL. Glycosylation of G-ePRL at Asn³¹ was confirmed by isolation and sequence analysis of an enzymatically derived G-ePRL glycopeptide spanning residues 29–37. Monosaccharide compositions of intact G-ePRL and this glycopeptide were very similar (Man₃, GlcNAc₂, GalNAc₁, Fuc_0.6, Gal_0.2, NeuAc_0.15) and resembled that of G-pPRL. The glycopeptide contained one sulfate residue as determined by ion chromatography after acid hydrolysis, indicating the presence of a sulfated monosaccharide. Comparative carbohydrate analysis of G-ePRL and other G-PRL preparations suggests that the functionally significant Asn³¹ carbohydrate unit is a fucosylated complex mono- and/or biantennary oligosaccharide terminating with a sulfated GalNAc residue and two or three Man residues.     

Visualization of embolism formation in the xylem of liana stems using neutron radiography

Background and Aims

Cold neutron radiography was applied to directly observe embolism in conduits of liana stems with the aim to evaluate the suitability of this method for studying embolism formation and repair. Potential advantages of this method are a principally non-invasive imaging approach with low energy dose compared with synchrotron X-ray radiation, a good spatial and temporal resolution, and the possibility to observe the entire volume of stem portions with a length of several centimetres at one time.

Methods

Complete and cut stems of Adenia lobata, Aristolochia macrophylla and Parthenocissus tricuspidata were radiographed at the neutron imaging facility CONRAD at the Helmholtz-Zentrum Berlin für Materialien und Energie, with each measurement cycle lasting several hours. Low attenuation gas spaces were separated from the high attenuation (water-containing) plant tissue using image processing.

Key results

Severe cuts into the stem were necessary to induce embolism. The formation and temporal course of an embolism event could then be successfully observed in individual conduits. It was found that complete emptying of a vessel with a diameter of 100 µm required a time interval of 4 min. Furthermore, dehydration of the whole stem section could be monitored via decreasing attenuation of the neutrons.

Conclusions

The results suggest that cold neutron radiography represents a useful tool for studying water relations in plant stems that has the potential to complement other non-invasive methods.     

Matrix metalloproteinase expression in cardiac myocytes following myocardial infarction in the rabbit

Myocardial infarction (MI), leads to cardiac remodeling, thinning of the ventricle wall, ventricular dilation, and heart failure, and is a leading cause of death. Interactions between the contractile elements of the cardiac myocytes and the extracellular matrix (ECM) help maintain myocyte alignment required for the structural and functional integrity of the heart. Following MI, reorganization of the ECM and the myocytes occurs, contributing to loss of heart function. In certain pathological circumstances, the ECM is modulated such that the structure of the tissue becomes damaged. The matrix metalloproteinases (MMPs) are a family of enzymes that degrade molecules of the ECM. The present experiments were performed to define the time-course, isozyme subtypes, and cellular source of increased MMP expression that occurs following MI in an experimental rabbit model. Heart tissue samples from infarcted and sham animals were analyzed over a time-course of 1-14 days. By zymography, it was demonstrated that, unlike the sham controls, MMP-9 expression was induced within 24 hours following MI. MMP-3 expression, also absent in sham controls, was induced 2 days after MI. MMP-2 expression was detected in both the sham and infarcted samples and was modestly up-regulated following MI. Tissue inhibitor of metalloproteinase-1 (TIMP-1) expression was evaluated and shown to be down-regulated following MI, inverse of MMP-9 and MMP-3 expression. Further, MMP-9 and MMP-3 expression was detected by immunohistochemistry in myocytes within the infarct. Additional studies were conducted in which cultured rat cardiac myocytes were exposed to a hypoxic environment (2% O2) for 24 hours and the media analyzed for MMP expression. MMP-9 and MMP-3 were induced following exposure to hypoxia. It is speculated that the net increase in proteolytic activity by myocytes is a contributing factor leading to myocyte misalignment and slippage. Additional studies with a MMP inhibitor would elucidate this hypothesis.     

Origin of the cytoplasmic pH changes during anaerobic stress in higher plant cells. Carbon-13 and phosphorous-31 nuclear magnetic resonance studies

We tested the contribution of nucleoside triphosphate (NTP) hydrolysis, ethanol, and organic acid syntheses, and H(+)-pump ATPases activity in the acidosis of anoxic sycamore (Acer pseudoplatanus) plant cells. Culture cells were chosen to alter NTP pools and fermentation with specific nutrient media (phosphate [Pi]-deprived and adenine- or glycerol-supplied). In vivo (31)P- and (13)C-nuclear magnetic resonance (NMR) spectroscopy was utilized to noninvasively measure intracellular pHs, Pi, phosphomonoesters, nucleotides, lactate, and ethanol. Following the onset of anoxia, cytoplasmic (cyt) pH (7.5) decreased to 6.8 within 4 to 5 min, whereas vacuolar pH (5.7) and external pH (6.5) remained stable. The NTP pool simultaneously decreased from 210 to <20 nmol g(-1) cell wet weight, whereas nuceloside diphosphate, nucleoside monophosphate, and cyt pH increased correspondingly. The initial cytoplasmic acidification was at a minimum in Pi-deprived cells containing little NTP, and at a maximum in adenine-incubated cells showing the highest NTP concentration. Our data show that the release of H(+) ions accompanying the Pi-liberating hydrolysis of NTP was the principal cause of the initial cyt pH drop and that this cytoplasmic acidosis was not overcome by H(+) extrusion. After 15 min of anoxia, a partial cyt-pH recovery observed in cells supplied with Glc, but not with glycerol, was attributed to the H(+)-consuming ATP synthesis accompanying ethanolic fermentation. Following re-oxygenation, the cyt pH recovered its initial value (7.5) within 2 to 3 min, whereas external pH decreased abruptly. We suggest that the H(+)-pumping ATPase located in the plasma membrane was blocked in anoxia and quickly reactivated after re-oxygenation.