Mutation proximal to the tRNA binding region of the Nicotiana plastid 16S rRNA confers resistance to spectinomycin.

Summary Nicotiana tabacum lines carrying maternally inherited resistance to spectinomycin were obtained by selection for green callus in cultures bleached by spectinomycin. Two levels of resistance was found. SPC1 and SPC2 seedlings are resistant to high levels (500 g/ml), SPC23 seedlings are resistant to low levels (50 g/ml) of spectinomycin. Lines SPC2 and SPC23 are derivatives of the SR1 streptomycin-resistant plastome mutant. Spectinomycin resistance is due to mutations in the plastid 16S ribosomal RNA: SPC1, an A to C change at position 1138; SPC2, a C to U change at position 1139; SPC23, a G to A change at position 1333. Mutations similar to those in the SPC1 and SPC2 lines have been previously described, and disrupt a conserved 16S ribosomal RNA stem structure. The mutation in the SPC23 line is the first reported case of a mutation close to the region of the 16S rRNA involved in the formation of the initiation complex. The new mutants provide markers for selecting plastid transformants.     

Nutrient levels in malformed and healthy tissues of mango (Mangifera indica L.)

Samples of malformed and healthy panicles of mango (Mangifera indica L.) as well as leaves and shoots bearing them were collected at different stages of development (fully swollen buds, bud inception, fully grown panicles prior to full bloom and at full bloom) over two consecutive years and were analysed for their macro- and micronutrient status. In addition, malformed and healthy seedlings were collected and analysed. Malformed panicles were found to be significantly higher in N at all the developmental stages except at bud inception. Phosphorus and K also tended to accumulate in malformed panicles at later stages of their development. In general, malformed panicles exhibited lower levels of P, K and Ca than healthy panicles. The differences in levels of Mg and S in malformed and healthy panicles were not significant. All micronutrients were in much lower concentrations in malformed panicles except for Mn which appears to accumulate in malformed panicles particularly at the early stages of development. The leaves on the shoots bearing malformed panicles also showed a tendency to accumulate N, while P, Mg and S were always higher in leaves on shoots bearing healthy panicles. The leaves on shoots bearing healthy panicles had lower levels of Fe, Cu and Mn, whereas levels of Zn and B tended to be higher in leaves on shoots bearing malformed panicles. The nutrient concentration differences between the two kinds of shoots were generally nonsignificant (P=0.05), except for K and S which were significantly lower in shoots bearing malformed panicles. The shoots bearing malformed panicles showed significantly (P=0.05) higher levels of almost all nutrients compared with shoots bearing healthy panicles. Vegetative malformation was found to be associated significantly (p=0.05) with higher amounts of all nutrients except Ca which was significantly higher in healthy seedlings. The present study, therefore, seems to point to lower Ca as one of the pre-disposing factors causing malformation in mango.A part of Ph.D. thesis of the senior author.A part of Ph.D. thesis of the senior author.     

Aminoglycoside-3″-adenyltransferase confers resistance to spectinomycin and streptomycin in Nicotiana tabacum

The bacterial gene aad A encodes the enzyme aminoglycoside-3-adenyltransferase that confers resistance to spectinomycin and streptomycin in Escherichia coli. Chimeric genes have been constructed for expression in plants, and were introduced into Nicotiana tabacum by Agrobacterium binary transformation vectors. Spectinomycin or streptomycin in selective concentrations prevent greening of N. tabacum calli. Transgenic clones, however, formed green calli on selective media containing spectinomycin, streptomycin, or both drugs. Resistance was inherited as a dominant Mendelian trait in the seed progeny. Resistance conferred by the chimeric aad A gene can be used as a color marker similar to the resistance conferred by the streptomycin phosphotransferase gene to streptomycin.     

Kanamycin resistance as a selectable marker for plastid transformation in tobacco

We report on a novel chimeric gene that confers kanamycin resistance on tobacco plastids. The kan gene from the bacterial transposon Tn5, encoding neomycin phosphotransferase (NPTII), was placed under control of plastid expression signals and cloned between rbcL and ORF512 plastid gene sequences to target the insertion of the chimeric gene into the plastid genome. Transforming plasmid pTNH32 DNA was introduced into tobacco leaves by the biolistic procedure, and plastid transformants were selected by their resistance to 50 g/ml of kanamycin monosulfate. The regenerated plants uniformly transmitted the transplastome to the maternal progeny. Resistant clones resulting from incorporation of the chimeric gene into the nuclear genome were also obtained. However, most of these could be eliminated by screening for resistance to high levels of kanamycin (500 g/ml). Incorporation of kan into the plastid genome led to its amplification to a high copy number, about 10000 per leaf cell, and accumulation of NPTII to about 1% of total cellular protein.     

Endothelium-dependent relaxation of rat aorta to a histamine H(3) agonist is reduced by inhibitors of nitric oxide synthase, guanylate cyclase and Na,K-ATPase

The possible involvement of different effector systems (nitric oxide synthase, guanylate cyclase, beta-adrenergic and muscarinic cholinergic receptors, cyclooxygenase and lipoxygenase, and Na(+),K(+)-ATPase) was evaluated in a histamine H(3) receptor agonist-induced ((R)alpha-methylhistamine, (R)alpha-MeHA) endothelium-dependent rat aorta relaxation assay. (R)alpha-MeHA (0.1 nM - 0.01 mM) relaxed endothelium-dependent rat aorta, with a pD(2) value of 8.22 +/- 0.06, compared with a pD(2) value of 7.98 +/- 0.02 caused by histamine (50% and 70% relaxation, respectively). The effect of (R)alpha-MeHA (0.1 nM - 0.01 mM) was competitively antagonized by thioperamide (1, 10 and 30 nM) (pA(2) = 9.21 +/- 0.40; slope = 1.03 +/- 0.35) but it was unaffected by pyrilamine (100 nM), cimetidine (1 muM), atropine (10 muM), propranolol (1 muM), indomethacin (10 muM) or nordthydroguaiaretic acid (0.1 mM). Inhibitors of nitric oxide synthase, L-N(G)-monomethylarginine (L-NMMA, 10 muM) and N(G)-nitro-L-arginine methylester (L-NOARG, 10 muM) inhibited the relaxation effect of (R)alpha-MeHA, by approximately 52% and 70%, respectively). This inhibitory effect of L-NMMA was partially reversed by L-arginine (10 muM). Methylene blue (10 muM) and ouabain (10 muM) inhibited relaxation (R)alpha-MeHA-induced by approximately 50% and 90%, respectively. The products of cyclooxygenase and lipoxygenase are not involved in (R)alpha-MeHA-induced endothelium-dependent rat aorta relaxation nor are the muscarinic cholinergic and beta-adrenergic receptors. The results also suggest the involvement of NO synthase, guanylate cyclase and Na(+),K(+)-ATPase in (R)alpha-MeHA-induced endothelium-dependent rat aorta relaxation.     

The evidence for histamine H(3) receptor-mediated endothelium-dependent relaxation in isolated rat aorta

The presence of histamine H(3) receptors was evaluated on the rat aorta endothelium. In the presence of pyrilamine (1 nM, 7 nM, 10 nM) or thioperamide (1 nM, 10 nM, 30 nM) the concentration-response curve for histamine-induced (0.1 nM - 0.01 mM) endothelium-dependent rat aorta relaxation was shifted to the right without significant change of the E(max) indicating competitive antagonism by pyrilamine (pA(2) = 9.33 +/- 0.34, slope = 1.09 +/- 0.36) or thioperamide (pA(2) =9.31 +/- 0.16, slope=0.94 +/- 0.10). Cimetidine (1 muM) did not influence histamine-induced endothelium-dependent rat aorta relaxation. In the presence of thioperamide (1 nM, 10 nM, 30 nM) the concentration-response curve for (R)alpha-MeHA-induced (0.1 nM - 0.01 mM) endothelium-dependent relaxation was shifted to the right without significant change of E(max) indicated competitive antagonism by thioperamide (pA(2) = 9.21 +/- 0.4, slope = 1.03 +/- 0.35). Pyrilamine (100 nM) or cimetidine (1 muM) did not influence (R)alpha-MeHA-induced endothelium-dependent rat aorta relaxation. These results suggest the presence of a heterogenous population of histamine receptors, H(1) and H(3), on rat aorta endothelium.     

Skeletal Manifestations of Hydatid Disease in Serbia: Demographic Distribution,Site Involvement,Radiological Findings,and Complications

Although Serbia is recognized as an endemic country for echinococcosis, no information about precise incidence in humans has been available. The aim of this study was to investigate the skeletal manifestations of hydatid disease in Serbia. This retrospective study was conducted by reviewing the medical database of Institute for Pathology (Faculty of Medicine in Belgrade), a reference institution for bone pathology in Serbia. We reported a total of 41 patients with bone cystic echinococcosis (CE) during the study period. The mean age of 41 patients was 40.9±18.8 years. In 39% of patients, the fracture line was the only visible radiological sign, followed by cyst and tumefaction. The spine was the most commonly involved skeletal site (55.8%), followed by the femur (18.6%), pelvis (13.9%), humerus (7.0%), rib (2.3%), and tibia (2.3%). Pain was the symptom in 41.5% of patients, while some patients demonstrated complications such as paraplegia (22.0%), pathologic fracture (48.8%), and scoliosis (9.8%). The pathological fracture most frequently affected the spine (75.0%) followed by the femur (20.0%) and tibia (5.0%). However, 19.5% of patients didn''t develop any complication or symptom. In this study, we showed that bone CE is not uncommon in Serbian population. As reported in the literature, therapy of bone CE is controversial and its results are poor. In order to improve the therapy outcome, early diagnosis, before symptoms and complications occur, can be contributive.     

A novel approach to plastid transformation utilizes the phiC31 phage integrase

Thus far plastid transformation in higher plants has been based on incorporation of foreign DNA in the plastid genome by the plastid's homologous recombination machinery. We report here an alternative approach that relies on integration of foreign DNA by the phiC31 phage site-specific integrase (INT) mediating recombination between bacterial and phage attachment sites (attB and attP, respectively). Plastid transformation by the new approach depends on the availability of a recipient line in which an attB site has been incorporated in the plastid genome by homologous recombination. Plastid transformation involves insertion of an attP vector into the attB site by INT and selection of transplastomic clones by selection for antibiotic resistance carried in the attP plastid vector. INT function was provided by either expression from a nuclear gene, which encoded a plastid-targeted INT, or expressing INT transiently from a non-integrating plasmid in plastids. Transformation was successful with both approaches using attP vectors with kanamycin resistance or spectinomycin resistance as the selective marker. Transformation efficiency in some of the stable nuclear INT lines was as high as 17 independently transformed lines per bombarded sample. As this system does not rely on the plastid's homologous recombination machinery, we expect that INT-based vectors will make plastid transformation a routine in species in which homologous recombination rarely yields transplastomic clones.