Variation in nitrate metabolism in biovars of Pseudomonas solanacearum

A collection of 327 strains of Pseudomonas solanacearum , representing five biovars, was divisible into three groups on the basis of differences in nitrate metabolism. Nine strains (2.8%), of which seven were biovar 2 from bacterial wilt of potato, were nitrate reduction-deficient and failed to produce nitrite from nitrate by either of two methods of detection in five different media. A second group of 231 strains, comprising biovars 1 and 2 and a single biovar 3 strain, produced nitrite from nitrate and grew vigorously in the presence of nitrate under anaerobic conditions but were deficient in ability to denitrify. A third group comprising 57 strains of biovar 3, 28 of biovar 4 and one each of biovar 2 and 5 produced nitrite from nitrate and gave profuse growth and gas production from nitrate under anaerobic conditions. However, production of gas from nitrate (denitrification) was not a consistently reproducible property in some of the media tested. Gas production results were most reproducible when a semi-solid succinate/nitrate or glycerol/nitrate medium was used. Serial passage of four nitrate reduction-deficient isolates in nitrate medium did not restore ability to reduce nitrate.     

Characterization of waldmeister, a novel developmental mutant in Arabidopsis thaliana

A novel Arabidopsis thaliana (L.) Heynh. developmental mutant,waldmeister (wam), is described. This mutant was found in theprogeny arising from an Ac-Ds tagging experiment, but does notappear to be tagged by an introduced transposon. This recessivenuclear mutation maps between GAPB and ap1 on chromosome 1 andshows extreme morphological and physiological changes in bothfloral and vegetative tissues. Changes to the vegetative phenotypeinclude altered leaf morphology, multiple rosettes, stem fasciation,retarded senescence and disturbed geotropic growth. Changesto the floral phenotype include delayed flowering, increasednumber of inflorescences, determinate inflorescences, alterednumber and morphology of floral organs, chimeric floral organs,and ectopic ovules . wam was crossed to a number of previouslydescribed floral mutants: apetela 2, apetela 3, pistillata,agamous, and leafy. The phenotype of the double mutant was ineach case additive. In the case of agamous, however, the indeterminaterepetitive floral structure of agamous was lacking, emphasizingthe determinate inflorescence growth of wam. The extreme phenotypeof the wam mutant is suggestive of a disturbance to a gene ofglobal importance in the regulation of plant growth and development. Key words: Arabidopsis thaliana, waldmeister, developmental mutant, flower mutant     

Influence of glycosylation inhibitors on dihydropyridine binding to cardiac cells

In primary cultures of neonatal rat heart cells we found a linear correlation between the number of L-type calcium channel-specific dihydropyridine (DHP) binding sites and spontaneous beating frequency (v).Formation of glycoproteins in tissue culture was suppressed by different inhibitors of N-glycosylation. This inhibition alters to a different extent the binding of the DHP ligand (+)-[methyl-³H]PN 200-110 and v. The most severe but reversible effect occurs at 6 g/ml tunicamycin (B_max 45% and v 6%, resp., of control), a slight increase in B_max at 0.1–0.5 mM castanospermine and 0.05–2.5 mM deoxymannojirimycin. The other inhibitors gave no significant alterations of B_max.     

Detachment of transformed cells

A parallel-plate flow chamber was used to quantify the detachment of normal cloned rat embryo fibroblasts (CREF) fibroblasts,ras-transformed CREF fibroblasts (CREF T24), and CREF T24 fibroblasts transfected with a Krev/RAP1A suppressor gene (HK B1) from a confluent monolayer of normal CREF fibroblasts to determine if the expression patterns of CD44 variants (mol wt 110 and 140 kDa) corresponded with detachment properties and metastatic potential. In the detachment assay, known shear stresses ranging from 20–24 dyn/cm² were applied to the adherent cells and the number of cells detached from the monolayer after 180 s was determined. Results showed that cellular expression of CD44 variants correlated with the metastatic potential of the cells and with the cells’ ability to detach from a monolayer of normal cells. Western blot analysis showed a low level of expression of the CD44 variants in the normal cell line, CREF, and the lowly metastatic cell line, HK B1. Detachment studies showed a low percentage of detachment of both of these cell lines from a normal cell monolayer. Tumor-derived (HK B1-T) and lung nodule-derived (HK B1-M) cell lines were established and both formed tumors and metastasis with reduced latency periods as compared to HK B1, but still showed a markedly delayed latency period compared to the highly metastatic cell line, CREF T24. Both of these cell lines showed a higher expression of the CD44 variants as compared to CREF and HK B1, and detached easier than CREF and HK B1. CREF T24 showed a much higher level of expression of the variants and had a higher percentage detachment than all other cell lines. To further test the role of the CD44 variants in the ability of the cells to detach from the normal monolayer, CREF cells were transfected with a DNA construct that constitutively expresses the CD44 variants and the detachment properties of three randomly selected clones were studied. Clones 2 and 3 showed a low level of expression of the CD44 variants after transfection and detached from the normal monolayer similar to CREF. Clone 1 showed a high level of expression of the CD44 variants and the detachment of these cells was significantly higher than CREF. From these results, it is concluded that in the five cell lines studied, expression of the CD44 variants play a significant role in the ability of the cells to detach from a monolayer of normal cells. It is hypothesized that this detachment may be an important component of a cell’s ability to metastasize.     

Transient expression in leaf mesophyll protoplasts of Arabidopsis thaliana

Conditions for maximising transient expression of GUS in leaf mesophyll protoplasts of Arabidopsis thaliana ecotype C24 were investigated. It was found that the factors most influencing expression levels, with optimum levels in parenthesis, were plasmid DNA quantity (100 g per 5 × 10⁵ protoplasts), inclusion of carrier DNA (50 g), PEG pH and amount (pH above 6, and total PEG concentration at least 9% w/w) and the topological form of the DNA. Linearised plasmid DNA with long flanking sequences 3 and 5 to the marker gene yielded the highest levels of GUS expression.Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid - GUS -glucuronidase - MU methylumbelliferone - PEG polyethylene glycol - X-gluc 5-bromo-4-chloro-3-indolyl--glucuronic acid     

Glycosylation in cardenolide biosynthesis

The glycosylation and deglycosylation of cardiac glycosides was investigated using cell suspension cultures and shoot cultures, both established from Digitalis lanata EHRH. plants, as well as isolated enzymes. Shoots were capable of glucosylating digitoxigenin, evatromonoside, digiproside, glucodigitoxigenin and digitoxin. Suspension cultured Digitalis cells glucosylated all the substrates mentioned but digiproside, whereas the UDP-glucosedependent cardinolide glucosyltransferase isolated from that source did not accept digitoxigenin and digiproside as substrates. It is concluded that at least three different glucosyltransferases are involved in cardiac glycoside formation in Digitalis. Similar experiments carried out with glucosylated cardenolides which were administered to cultured cells, shoots and a cardenolide -glucosidase isolated from young leaves revealed that at least two different glucosidases occur in Digitalis lanata, albeit in different tissues or during different phases of development. The biotransformation of glucoevatromonoside was investigated using unlabelled compound and [¹⁴C-glucose]-glucoevatromonoside synthesized enzymatically. After 7 d of incubation almost no radioactivity could be recovered from the cardenolide fraction, indicating that the terminal glucose of glucoevatromonoside was now incorporated into volatile, hydrophilic and insoluble compounds. Since, on the other hand, large amounts of cardenolides were found in the experiments with unlabelled glucoevatromonoside it is assumed that steady state or pool size regulation is achieved by the coordinated action of a cardenolide glucosidase and a glucosyltransferase.Abbreviations Acdox D-acetyldigitoxose - dgen digoxigenin - dox D-digitoxose - dten digitoxigenin - dtl D-digitalose - fuc D-fucose - gten gitoxigenin - qun D-quinovose - CGH cardenolide 16-O-glucohydrolase - DFT UDP-fucose:digitoxigenin 3-O-fucosyltransferase - DGT UDP-glucose:Digitoxin 16-O-glucosyltransferase - DQT UDP-quinovose:digitoxigenin 3-O-quinovosyltransferase     

A mating-type factors of Coprinus cinereus have variable numbers of specificity genes encoding two classes of homeodomain proteins

We have identified the seven genes that constitute the A43 mating-type factor of Coprinus cinereus and compare the organisation of A43 with the previously characterised A42 factor. In both, the genes that trigger clamp cell development, the so-called specificity genes, are separated into and loci by 7 kb of noncoding sequence and are flanked by homologous genes -fg and -fg. The specificity genes are known to encode two classes of dissimilar homeodomain (HD1 and HD2) proteins and have different allelic forms which show little or no cross-hybridisation. By partial sequencing we identified a divergently transcribed HD1 (a1-2) and HD2 (a2-2) gene in the A43 locus. a2-2 failed to elicit clamp cell development in three different hosts, suggesting that it is non-functional. a1-2 elicited clamp cells in an A42 host that has only an HD2 gene (a2-1) in its locus, thus demonstrating that the compatible A mating interaction is between an HD1 and an HD2 protein. The A43 locus contains three specificity genes, the divergently transcribed HD1 and HD2 genes b1-2 and b2-2 and a third HD1 gene (d1-1) that was shown by hybridisation and transformation analyses to be functionally equivalent to d1-1 in A42. An untranscribed footprint of a third A42 HD1 gene, c1-1, was detected between the A43 b2-2 and d1-1 genes by Southern hybridisation.     

Histological study of pancreatic beta-cell loss in relation to the insulitis process in the non-obese diabetic mouse

Non-obese diabetic (NOD) mice spontaneously develop an autoimmune diabetes with higher incidence in females than in males. In order to elucidate possible factors involved in the different incidence of diabetes between male and female mice, we studied the progression of pancreatic beta-cell loss in relation to mononuclear cell infiltration of the pancreas (insulitis). We examined the pancreas of 76 NOD mice (39 males and 37 females) of different ages. The beta-cell content was assessed by immunoperoxidase staining of sections with an anti-insulin serum and the severity of insulitis was determined by haematoxylin staining of the same sections. A semi-quantitative criterion was used to grade both parameters. The results showed that females have a faster loss of beta-cell mass, which progressively decreases with the increase of severity of insulitis. In males, a medium to severe degree of insulitis is required before initial loss of beta cells occurs. Under the age of 10 weeks there was a significantly lower content of beta cells in females than males (2.84±0.03 vs 2.67±0.07; P=0.02). Since we never observed a significant difference in the degree of mononuclear cell infiltration in age-matched males and females, these data support the hypothesis of weaker beta-cell resistance to immunological attack in female mice. Thus beta-cell sensitivity, in addition to immunological activity, is an important factor in the pathogenesis of insulin dependent diabetes.     

Analysis of a primer-independent GTF-I from Streptococcus salivarius

Abstract A glucosyltransferase (GTF) gene, designated gtfL , from Streptococcus salivarius was cloned and expressed in Escherichia coli and its nucleotide sequence determined. The GTF-L enzyme catalysed the synthesis of water-insoluble glucan in a primer-independent manner. The nucleotide sequence and derived amino acid sequence of GTF-L were similar in size and domain structure to previously sequenced glucosyltransferases. However, a 464-bp region of high variability was identified which could be selectively amplified from strains of S. salivarius by the polymerase chain reaction and could therefore form the basis for species identification. No sequence-specific motifs related to the solubility and linkage of the glucan product or its need for a dextran primer could be ascertained.     

The first decade of oligotrophication of Lake Constance

In Lake Constance, after several decades of cutrophication, a decrease in phosphorus loading over the last decade has lead to a partial recovery from eutrophication. Here we analyse the shift in the taxonomic composition of phytoplankton during the first decade of oligotrophication in Lake Constance. During the 1980s, spring total P concentrations decreased from ca. 130 to less than 50 ·l^–1. This decrease was reflected by an approximately proportional decrease in summer phytoplankton biomass while spring phytoplankton biomass seemed unresponsive. Major taxonomic changes occured during both growth seasons. In spring, the proportion of diatoms, green algae and Chrysophyta increased while the proportion of Cryptophyta decreased. The summer trend was very different: the relative importance of diatoms decreased and Cryptophyta and Chrysophyta increased, while Chlorophyta reached their peak around 1985. These trends are also analysed at the genus level. Comparison with taxonomic trends during the eutrophication period shows the expected reversals in most cases. Comparison with other lakes shows general similarities, with the notable exception that Planktothrix rubescens has never been important in Lake Constance. The increase of diatoms during spring is attributed to their improved competitive performance with increasing Si:P ratios. Their decrease during summer is explained by the increasing silicate removal from the epilimnion by increasing spring populations.