DNA fingerprinting detects genetic variability in the pearl millet downy mildew pathogen (Sclerospora graminicola)

Genetic variability in six host genotype-specific pathotypes of pearl millet downy mildew pathogen S. graminicola was studied at the molecular level using mini- and micro-satellites. Our results indicated that microsatellites (GAA)₆, (GACA)₄, and especially (GATA)₄ were quite informative and showed high levels of polymorphism among the pathotypes. The six pathotypes could be classified into five groups based on the cluster analysis of their genetic similarities, thereby confirming the existence of distinct host genotype-specific virulence in S. graminicola pathotypes. We demonstrate, for the first time, the use of DNA fingerprinting to detect genetic variation in downy mildew fungus of pearl millet.     

Growth and monoterpene production by transformed shoot cultures of Mentha citrata and Mentha piperita in flasks and fermenters

This paper reports studies on the growth and biosynthesis of monoterpenes by transformed shoot cultures of Mentha citrata and Mentha piperita, originally developed 5 years ago and since maintained by regular subculturing. Throughout this time, the M. citrata culture has stably maintained production of an oil closely resembling that of the parent plant in which linalool and linalyl acetate are the predominant components. However, M. piperita, which initially showed a divergence from the parent plant in producing significant amounts of menthofuran in addition to the characteristic oil components menthol and menthone, has now been found to produce pulegone and menthofuran as the major components. The cultures were subjected to different environmental conditions of varying periods of light and temperature in an attempt to restore menthol and menthone production. Increased illumination reduced the yields of pulegone and menthofuran but did not stimulate the production of either menthol or menthone, which remained only at trace levels (below 0.2 g/g fresh weight). Cultures of M. citrata were, however, stimulated by increased illumination, and produced more linalool and linalyl acetate. Shoot cultures of M. citrata and M. piperita were grown in 14–1 fermenters for up to 60 dys during which the biomass increased from approximately 100 g to 2.5 kg and 3.5 kg respectively. Both cultures rapidly consumed sucrose with a concomitant release of glucose, and the uptake of inorganic ions was similar except that M. citrata consumed far less Na+ during the fermentation. The total yields of monoterpenes from the fermentations were 1.16 g (M. piperita) and 0.18 g (M. citrata). *** DIRECT SUPPORT *** AG903062 00005     

Superoxide dismutase activity of the captopril-iron complex

With an assay that generates superoxide anion radicals without the intervention of metal ions we investigated the antioxidant properties of captopril, an angiotensin-converting enzyme inhibitor with a sulfhydryl group. Under these conditions, increasing concentrations of the drug were seen not to scavenge O· ₂ ^– directly. However, a combination of captopril and iron could bring about the breakdown of the superoxide anion; a result that may help to understand the free radical-scavenging properties of captopril.     

Mammalian-heart adenylate deaminase: Cross-species immunoanalysis of tissue distribution with a cardiac-directed antibody

A sheep antiserum against purified rabbit-heart adenylate deaminase (EC 3.5.4.6) (AMPD) was developed and validated as an immunologic probe to assess the cross-species tissue distribution of the mammalian cardiac AMPD isoform. The antiserum and the antibodies purified therefrom recognized both native and denatured rabbit-heart AMPD in immunoprecipitation and immunoblot experiments, respectively, and antibody binding did not affect native enzyme activity. The immunoprecipitation experiments further demonstrated a high antiserum titer. Immunoblot analysis of either crude rabbit-heart extracts or purified rabbit-heart AMPD revealed a major immunoreactive band with the molecular mass (81 kDa) of the soluble rabbit-heart AMPD subunit. AMPD in heart extracts from mammalian species other than rabbit (including human) was equally immunoreactive with this antiserum by quantitative immunoblot criteria. Although generally held to be in the same isoform class as heart AMPD, erythrocyte AMPD was not immunoreactive either within or across species. Nor was AMPD from most other tissues [e.g., white (gastrocnemius) muscle, lung, kidney] immunoreactive with the cardiac-directed antibody. Limited immunoreactivity was evidenced by mammalian liver, red (soleus) muscle, and brain extracts across species, indicating the presence of a minor cardiac(-like) AMPD isoform in these tissues. The results of this study characterize the tissue distribution of the cardiac AMPD isoform using a molecular approach with the first polyclonal antibodies prepared against homogeneous cardiac AMPD. This immunologic probe should prove useful at the tissue level for AMPD immunohistochemistry.     

Regulation of phospholamban and troponin-I phosphorylation in the intact rat cardiomyocytes by adrenergic and cholinergic stimuli: roles of cyclic nucleotides,calcium, protein kinases and phosphatases and depolarization

Protein phosphorylation was investigated in [³²P]-labeled cardiomyocytes isolated from adult rat heart ventricles. The -adrenergic stimulation (by isoproterenol, ISO) increased the phosphorylation of inhibitory subunit of troponin (TN-I), C-protein and phospholamban (PLN). Such stimulation was largely mediated by increased adenylyl cyclase (AC) activity, increased myoplasmic cyclic AMP and increased cyclic AMP dependent protein kinase (A-kinase)-catalyzed phosphorylation of these proteins in view of the following observations: (a) dibutyryl-and bromo-derivatives of cyclic AMP mimicked the stimulatory effect of ISO on protein phosphorylation while (b) Rp-cyclic AMP was found to attenuate ISO-dependent stimulation. Unexpectedly, 8-bromo cyclic GMP was found to markedly increase TN-I and PLN phosphorylation. Both ₁- and ₂-adrenoceptors were present and ISO binding to either receptor was found to stimulate myocyte AC. However, the stimulation of the ₂-AR only marginally increased while the stimulation of ₁-AR markedly increased PLN phosphorylation. Other stimuli that increase tissue cyclic AMP levels also increased PLN and TN-I phosphorylation and these included isobutylmethylxanthine (non-specific phosphodiesterase inhibitor), milrinone (inhibits cardiotonic inhibitable phosphodiesterase, sometimes called type III or IV) and forskolin (which directly stimulates adenylyl cyclase). Cholinergic agonists acting on cardiomyocyte M₂-muscarinic receptors that are coupled to AC via pertussis toxin(PT)-sensitive G proteins inhibited AC and attenuated ISO-dependent increases in PLN and TN-I phosphorylation. Thein vivo PT treatment, which ADP-ribosylated G_i-like protein(s) in the myocytes, markedly attenuated muscarinic inhibitory effect on PLN and TN-I phosphorylation on one hand and, increased the -adrenergic stimulation, on the other. Controlled exposure of isolated myocytes to N-ethyl maleimide, also led to the findings similar to those seen following the PT treatment. Exposure of myocytes to phorbol, 12-myristate, 13-acetate (PMA) increased the protein phosphorylation, augmenting the stimulation by ISO, and such augmentation was antagonized by propranolol suggesting modulation of the -adrenoceptor coupled AC pathway by PMA. Okadaic acid (OA) exposure of myocytes also increased protein phosphorylation with the results supporting the roles for type 1 and 2A protein phosphatases in the dephosphorylation of PLN and TN-I. Interestingly OA treatment attenuated the muscarinic inhibitory effect which was restored by subsequent brief exposure of myocytes to PMA. While the stimulation of alpha adrenoceptors exerted little effect on the phosphorylation of PLN and TN-I, inactivation of alpha adrenoceptors by chloroethylclonidine (CEC), augmented -adrenergically stimulated phosphorylation. KCl-dependent depolarization of myocytes was observed to potentiate ISO-dependent increase in phosphorylation (incubation period 15 sec to 1 min) as well as to accelerate the time-dependent decline in this phosphorylation seen upon longer incubation. Verapamil decreased ISO-stimulated protein phosphorylation in the depolarized myocytes. Depolarization was found to have little effect on the muscarinic inhibitory action on phosphorylation. Prior treatment of myocytes with PMA, was found to augment ISO-stimulated protein phosphorylation in the depolarized myocytes. Such augmented increases were completely blocked by propranolol. Forskolin also stimulated PLN and TN-I phosphorylation. Prior exposure of myocytes to forskolin followed by incubation in the depolarized and polarized media showed that PLN was dephosphorylated more rapidly in the depolarized myocytes. The results support the view that both cyclic AMP and calcium signals cooperatively increase the rates of phosphorylation of TN-I and PLN in the depolarized cardiomyocytes during -adrenergic stimulation. The results raise the additional possibility that the calcium signal may regulate the dephosphorylation of PLN in the depolarized cell. While muscarinic attenuation of -adrenergic action on protein phosphorylation was mediated, in part, by decreased AC activity, and muscarinic inhibition of AC and protein phosphorylation was not detectably influenced by the depolarization, the evidence was seen that muscarinic stimulation of dephosphorylation mechanisms are intimately involved. The postulate that the simultaneous stimulation of ₁-adrenoceptors inhibits -adrenergic stimulation of PLN and TN-I phosphorylation is supported.     

Isolation, Purification and Partial Characterisation of Urease from Seeds of Water Melon (Citrullus vulgaris)

Urease from seeds of water melon was purified to apparent homogeniety upto a sp act of 3750 units/mg protein with 31% recovery. Enzyme showed single protein band on native PAGE by urease specific staining. The mol wt of the enzyme was 4,70,000 and the preparation was free from bound nucleotides (A₂₈₀/A₂₆₀=1.14). The enzyme exhibited maximum activity in 50 mM Tris-acetate buffer (pH 8.5). The Km for urease was 8 mM. The enzyme was not inhibited by 25 mM of EDTA in 50 mM Tris-acetate buffer (pH 8.0 and 8.5).     

Aggregation of β-Amyloid Peptide Is Promoted by Membrane Phospholipid Metabolites Elevated in Alzheimer's Disease Brain

Abstract: Increased amounts of β-amyloid (Aβ) peptide deposits are found in Alzheimer's disease brain. These amyloid deposits have been implicated in the pathophysiology of this common dementing illness. Aβ peptides have been shown to be toxic to neurons in cell culture, and this toxicity is critically dependent on the aggregation of the peptide into cross-β-pleated sheet fibrils. Also, in vivo and postmortem NMR studies have shown changes in certain brain membrane phospholipid metabolites in normal aging and more extensive alterations in patients with Alzheimer's disease. The finding that membrane phospholipids affect the aggregation of Aβ suggests that the abnormalities in membrane metabolism found in Alzheimer's disease could affect the deposition of Aβ in vivo. Therefore, we examined the effect of membrane phospholipid metabolites that are altered in Alzheimer's disease brain on the aggregation of Aβ(1–40) using a light scattering method. Certain metabolites (glycerophosphocholine, glycerophosphoethanolamine, and α-glycerophosphate) augment the aggregation of Aβ. Other membrane phospholipid metabolites (phosphocholine, phosphoethanolamine, and inositol-1-phosphate) have no effect. We conclude that increased membrane phospholipid metabolite concentrations may play a role in the deposition of Aβ seen in normal aging and the even greater deposition of Aβ observed in Alzheimer's disease.     

Human immunodeficiency virus-associated vasculopathy in transgenic mice.

There is substantial clinical evidence for the development of vascular disorders in human immunodeficiency virus (HIV)-infected individuals, particularly in the form of vasculitis. Transgenic mice carrying a replication-defective HIV-1 provirus with selective deletion of the gag, pol, and env genes developed extensive vasculopathy. Restricted expression of HIV nonstructural genes in smooth muscle cells was accompanied by the migration and proliferation of these cells in blood vessels of all sizes and at different body sites. The frequent infiltration observed in the hypertrophic vessel walls occurred predominantly in the adventitia and was composed of primarily T cells and occasionally plasma cells. The intimal thickening generated significant luminal narrowing in some vessels, and the restricted blood flow led to ischemia in the affected tissues. Interestingly, the endothelium did not appear to support HIV gene expression or be involved in the pathological process. This transgenic model provides an opportunity to dissect the mechanism underlying HIV-associated vasculopathy.