Detection of Ralstonia solanacearum, which causes brown rot of potato, by fluorescent in situ hybridization with 23S rRNA-targeted probes

During the past few years, Ralstonia (Pseudomonas) solanacearum race 3, biovar 2, was repeatedly found in potatoes in Western Europe. To detect this bacterium in potato tissue samples, we developed a method based on fluorescent in situ hybridization (FISH). The nearly complete genes encoding 23S rRNA of five R. solanacearum strains and one Ralstonia pickettii strain were PCR amplified, sequenced, and analyzed by sequence alignment. This resulted in the construction of an unrooted tree and supported previous conclusions based on 16S rRNA sequence comparison in which R. solanacearum strains are subdivided into two clusters. Based on the alignments, two specific probes, RSOLA and RSOLB, were designed for R. solanacearum and the closely related Ralstonia syzygii and blood disease bacterium. The specificity of the probes was demonstrated by dot blot hybridization with RNA extracted from 88 bacterial strains. Probe RSOLB was successfully applied in FISH detection with pure cultures and potato tissue samples, showing a strong fluorescent signal. Unexpectedly, probe RSOLA gave a less intense signal with target cells. Potato samples are currently screened by indirect immunofluorescence (IIF). By simultaneously applying IIF and the developed specific FISH, two independent targets for identification of R. solanacearum are combined, resulting in a rapid (1-day), accurate identification of the undesired pathogen. The significance of the method was validated by detecting the pathogen in soil and water samples and root tissue of the weed host Solanum dulcamara (bittersweet) in contaminated areas.     

Genetic characterization of RRS1, a recessive locus in Arabidopsis thaliana that confers resistance to the bacterial soilborne pathogen Ralstonia solanacearum

The soilborne, vascular pathogen Ralstonia solanacearum, the causative agent of bacterial wilt, was shown to infect a range of Arabidopsis thaliana accessions. The pathogen was capable of infecting the Col-5 accession in an hrp-dependent manner, following root inoculation. Elevated bacterial population levels were found in leaves of Col-5, 4 to 5 days after root inoculation by the GMI1000 strain. Bacteria were found predominantly in the xylem vessels and spread systematically throughout the plant. The Nd-1 accession of A. thaliana was resistant to the GMI1000 strain of R. solanacearum. Bacterial concentrations detected in leaves of Nd-1, inoculated with an hrp+ strain of R. solanacearum, were only slightly higher than those detected in the susceptible accession, Col-5, following inoculation with a strain whose hrp gene cluster was deleted. Leaf inoculation of the GMI1000 strain on the resistant accession Nd-1 induced the formation of lesions in the older leaves of the rosette whereas the same strain of R. solanacearum provoked complete wilting of Col-5. Resistance to strain GMI1000 of R. solanacearum segregated as a simply inherited recessive trait in a genetic cross between Col-5 and Nd-1. F9 recombinant inbred lines generated between these two accessions were used to map a locus, RRS1, that was the major determinant of resistance between restriction fragment length polymorphism markers mi83 and mi61 on chromosome V. This region of the A. thaliana genome is known to contain many other pathogen recognition capabilities.     

Transfer RNA mimicry in a new group of positive-strand RNA plant viruses, the furoviruses: differential aminoacylation between the RNA components of one genome

The effects of the organic calcium channel blocker verapamil and the beta-receptor blocker propranolol on dipole (phi(d)) and surface (phi(s)) potentials of bilayer lipid membranes were studied. The boundary potentials (phi(b)= phi(d) + phi(s)) of black lipid membranes, monitored by conductance measurements in the presence of nonactin and by capacitive current measurements were compared with phi(s) calculated from the electrophoretic mobility of lipid vesicles. It was shown that the increase of boundary potential, induced by the adsorption of the positively charged propranolol, was caused solely by an increase in surface potential. Although phi(s) also increases due to the adsorption of verapamil, phi(b) diminishes. A sharp decrease of the dipole potential was shown to be responsible for this effect. From Langmuir adsorption isotherm the dissociation constant Kd of verapamil was estimated. The uncharged form of verapamil (Kd=(0.061+/-0.01) mM at pH 10.5) has a tenfold higher affinity to a neutral bilayer membrane than the positively charged form. The alteration of membrane dipole potential due to verapamil adsorption may have important implications for both membrane translocation and partitioning of small or hydrophobic ions and charged groups of membrane proteins.     

A new marker for mosaic analysis in Caenorhabditis elegans indicates a fusion between hyp6 and hyp7, two major components of the hypodermis

A fusion of the sur-5 protein to the green fluorescent protein containing a nuclear localization signal is demonstrated as a marker for genetic mosaic analysis in the nematode Caenorhabditis elegans. Because of an extensive accumulation of bright fluorescence in many nuclei, normal growth plates, each containing hundreds of worms, can be rapidly screened with a dissecting microscope for rare mosaic individuals. As the marker can also be used to detect transgenic worms, the construction of strains for mosaic analyses can be minimized. In the course of examining rare mosaic animals, an unexpected pattern of fluorescence was noticed for hyp6, a syncytial component of the hypodermis, which indicated that the marker may serve as a means of assessing cellular fusions during development. Immunofluorescent staining of adherens junctions confirmed a postembryonic fusion of hyp6 with hyp7, the major syncytium of the hypodermis.     

Pardaxin, a new pharmacological tool to stimulate the arachidonic acid cascade in PC12 cells

BACKGROUND: Calcium channel blockers (CCBs) are known to have differential effects on both changes in proteinuria as well as progression of diabetic nephropathy. No clinical study, however, has evaluated whether the differential antiproteinuric effects of CCBs may be explained by their effect on glomerular membrane permeability. We, therefore, tested the hypothesis that certain subclasses of CCBs reduce proteinuria by changing size selectivity of the glomerular membrane, hence changing its permeability. METHODS: Twenty-one patients with type 2 diabetes and the presence of nephropathy with hypertension were randomized to receive either diltiazem CD or nifedipine GITS after baseline data for mean systolic and diastolic pressure, urinary protein excretion, glomerular filtration rate, renal plasma flow, neutral dextran and IgG clearances were obtained. Glomerular filtration rate, renal plasma flow, neutral dextran and IgG clearance were measured every three months, arterial pressure and heart rate every month. Patients were followed for 21 months. RESULTS: At 21 months, both patient groups had similar levels of blood pressure control, however, only the diltiazem group had a change in proteinuria (4+/-10%delta, nifedipine vs. -57+/-18%delta, diltiazem; P < 0.001) with improvement in glomerular size selectivity and change in IgG clearance. CONCLUSIONS: These data support the hypothesis that CCBs that provide sustained reductions in proteinuria do so, in part, by improving glomerular size permselectivity.     

Toxicity and toxic components of two xanthid crabs, Atergatis floridus and Demania reynaudi, in Taiwan

Paralytic toxicity was detected by tetrodotoxin bioassay in eight specimens of Atergatis floridus and seven specimens of Demania reynaudi, collected from Taiwan in 1994. The toxicity of crab specimens was 161 +/- 115 (mean +/- S.D.) mouse units (MU) for A. floridus and 640 +/- 273 MU for D. reynaudi. The respective toxins were partially purified from specimens of A. floridus and D. reynaudi by ultrafiltration using a Diaflo YM-1 membrane, followed by chromatography on a Bio-Gel P-2 column. Electrophoresis, thin-layer chromatography, high-performance liquid chromatography, ultraviolet spectrum and gas chromatography-mass spectrometry indicated that the toxin of A. floridus was mainly composed of tetrodotoxin (85%), along with minor gonyautoxin 1-4 (15%), and the toxin of D. reynaudi was mainly composed of tetrodotoxin (88%), along with minor gonyautoxin 2-4 and neosaxitoxin (12%).     

A nonisothermal method of investigating the sintering kinetics of materials controlled by two mechanisms

Conclusions A nonisothermal method has been developed for determining the kinetic parameters of a materials sintering process controlled by two mechanisms. Using this method, it is possible to obtain the required information by performing two nonisothermal experiments at different specimen heating rates. The method has been employed for analyzing the kinetics of activated sintering of corundum.Translated from Poroshkovaya Metallurgiya, No. 7(163), pp. 36–41, July, 1976.     

Substrate induction and glucose repression of maltose utilization by Streptomyces coelicolor A3(2) is controlled by malR, a member of the lacl-galR family of regulatory genes

The rat treated with bile duct ligation (BDL) and furan is a unique animal model of massive bile ductular hyperplasia in which normal liver parenchyma is largely replaced with well-differentiated proliferated bile ductules. We have now developed a simple cell isolation procedure to obtain and culture viable bile ductular epithelial cells in high numbers and with a high degree of purity from the livers of BDL/furan-treated rats. Primary monolayer cell cultures were readily established when the isolated bile ductular epithelial cells were cultured in plastic tissue culture wells coated with rat tail tendon type I collagen plus bovine plasma fibronectin. Under these conditions, epidermal growth factor (EGF) was mitogenic for the cultured cells, and they retained phenotypic features typical of hyperplastic bile ductular epithelium but did not show evidence of ductal morphogenesis in vitro. In contrast, when the isolated bile ductular cells were cultured for 7 to 16 days in the presence of 25 ng EGF/mL and 10% fetal bovine serum on type I collagen gels, they formed into branching ductal structures whose ultrastructural features very closely resembled those of polarized hyperplastic bile ductules/ducts in vivo. Histological preparations of these gel cultures further showed that numerous ductal structures with defined lumens were present. Phenotypically, these ductal structures were completely surrounded by a thickened basement membrane that was strongly immunoreactive for laminin. Like their in vivo biliary cell counterparts, the epithelial cells comprising these ductal structures in culture also exhibited strong immunocytochemical staining reactions for cytokeratins 8 and 19, for glutathione S-transferase pi 7, and for luminal gamma-glutamyl transpeptidase, but they did not express immunoreactive albumin or alpha-fetoprotein. Occasional epithelial cells of the ductal structures, when examined in 10-day-old primary gel culture, showed strong nuclear staining for incorporated 5-bromo-2'deoxyuridine, indicating active cell proliferation. Our results support the development of a novel biliary epithelial cell culture model that has the potential of serving as a powerful tool for investigation of factors that regulate hyperplastic bile ductular morphogenesis, cell proliferation, and polarized cell functions in a structural form in vitro that mimics that of hyperplastic bile ductules induced in vivo.     

Retention in situ and spectral analysis of fluorescent vacuole components in sections of plant tissues

This paper describes an educational programme where clinical guidelines are used in dental education and assesses the value of these guidelines as perceived by undergraduate students. It presents a comparison of students' own assessments of their performance with those of their instructors, according to such guidelines. The educational programme at the Faculty of Odontology, Lund University, is described through 3 main principles: problem-based learning, adoption of a holistic attitude to patient care and the promotion of oral health. The dental curriculum occupies 5 years comprising 10 semesters. 41, 5th semester students (16 male and 25 female) participated in the study. Students were introduced to guidelines in group discussions at the beginning of the semester. At the completion of a patient's planned course of treatment, the outcome was formally assessed both by students and clinical instructors. Performance was scored in various categories as "excellent", "acceptable" or "unacceptable" according to the standards defined. A total of 1373 scores were made by both students and instructors. 88% cent of students used the guidelines often or almost always and found them useful. Overall, no students received an "unacceptable" score and 40% of students achieved a score of "excellent". Female students tended towards a higher score, but this was not significant. Instructors' and students' scores agreed in almost 90% of instances. Students under-scored their performance more frequently than they over-scored it. It appears that the use of clinical guidelines may encourage an increased awareness of the decision-making processes involved in clinical practice, but it must be acknowledged that these guidelines are complex both in derivation and application.     

Different genetic components in the Ethiopian population, identified by mtDNA and Y-chromosome polymorphisms

Structural equation modeling procedures were used to examine relationships among several war zone stressor dimensions, resilience-recovery factors, and post-traumatic stress disorder symptoms in a national sample of 1,632 Vietnam veterans (26% women and 74% men). A 9-factor measurement model was specified on a mixed-gender subsample of the data and then replicated on separate subsamples of female and male veterans. For both genders, the structural models supported strong mediation effects for the intrapersonal resource characteristic of hardiness, postwar structural and functional social support, and additional negative life events in the postwar period. Support for moderator effects or buffering in terms of interactions between war zone stressor level and resilience-recovery factors was minimal.     

A new family of lipolytic plant enzymes with members in rice, arabidopsis and maize

We have noted a striking similarity between the sequences of proteins in a novel family of lipases we recently reported [Upton, C. and Buckley, J. T. (1995) Trends Biol. Sci. 20, 178-9] and more than 120 sequences from the database of Expressed Sequence Tags (dbEST) which correspond to at least 30 unique genes from arabidopsis, rice and maize. A cDNA (Arab-1) corresponding to one of these sequences was isolated, sequenced and translated. There was significant similarity to sequences in the new lipase family over the entire open reading frame of Arab-1 and when expressed in E. coli, the gene product was lipolytic. Arab-1 and genes for some of the other plant proteins appear to be differentially expressed. They may play a role in the regulation of lipid metabolism during plant development.