Gating of water channels (aquaporins) in cortical cells of young corn roots by mechanical stimuli (pressure pulses): effects of ABA and of HgCl2

Hydraulic properties (half-time of water exchange, T1/2, and hydraulic conductivity, Lp; T1/2 approximately 1/Lp) of individual cells in the cortex of young corn roots were measured using a cell pressure probe for up to 6 h to avoid variations between cells. When pulses of turgor pressure of different size were imposed, T1/2 (Lp) responded differently depending on the size. Pulses of smaller than 0.1 MPa, which induced a small proportional water flow, caused no changes in T1/2 (Lp). Medium-sized pulses of between 0.1 and 0.2 MPa caused an increase in T1/2 (decrease in Lp) by a factor of 4 to 23. The effects caused by medium-sized pulses were reversible within 5-20 min. When larger pulses of more than 0.2 MPa were employed, changes were not reversible within 1-3 h, but could be reversed within 30 min in the presence of 500 nM of the stress hormone ABA. Cells with a short T1/2 responded to the aquaporin blocker mercuric chloride (HgCl2). The treatment had no effect on cells which exhibited long T1/2 following a mechanical inhibition by the large-pulse treatment. Step decreases in pressure resulted in the same inhibition as step increases. Hence, the treatment did not cause a stretch-inhibition of water channels and was independent of the directions of both pressure changes and water flows induced by them. It is concluded that inhibition is caused by the absolute value of intensities of water flow within the channels, which increased in proportion to the size of step changes in pressure. Probable mechanisms by which the mechanical stimuli are perceived are (i) the input of kinetic energy to the channel constriction (NPA motif of aquaporin) which may cause a conformational change of the channel protein (energy-input model) or (ii) the creation of tensions at the constriction analogous to Bernoulli's principle for macroscopic pores (cohesion-tension model). Estimated rates of water flow within the pores were a few hundred micro m s-1, which is too small to create sufficient tension. They were much smaller than those proposed for AQP1. Based on literature data of single-channel permeability of AQP1, a per channel energy input of 200 kBxT (kB=Boltzmann constant) was estimated for the energy-input model. This should be sufficient to initiate changes of protein conformation and an inactivation of channels. The data indicate different closed states which differ in the amount of distortion and the rates at which they relax back to the open state.     

Abscisic acid (ABA) flows from Hordeum vulgare to the hemiparasite Rhinanthus minor and the influence of infection on host and parasite abscisic acid relations

Using the facultative root hemiparasite Rhinanthus minor and Hordeum vulgare as a host, the flows, depositions, and metabolism of abscisic acid (ABA) within the host, within the parasite, and between host and parasite have been studied. When the plants were supplied with 5 mM NO(3)(-), there were weak or no effects of parasitism on ABA flows, biosynthesis, and ABA degradation in barley. However, ABA deposition was significantly affected in the leaf laminae (3-fold) and in the leaf sheath (2.4-fold), but not in roots. Dramatic changes in ABA flows, metabolism, and deposition on a per plant basis, however, have been observed in Rhinanthus. Biosynthesis in the roots was 12-fold higher after attachment, resulting in 14-fold higher ABA flows in the xylem. A large portion of this ABA was metabolized, a small portion was deposited. Phloem flows of ABA were increased 13-fold after attachment. The concentrations of ABA in tissues and transport fluids were higher in attached Rhinanthus by an order of magnitude than in host tissues and xylem sap. The same tendency was also found in a comparison between single Rhinanthus and unparasitized barley. As compared with 5 mM NO(3)(-), lower NO(3)(-) or 1 mM NH(4)(+) supply doubled the ABA concentrations in barley leaf laminae, while having only small or non-significant effects in the other organs. The possible function of ABA for the parasite is discussed.     

Effect of Osmotic Stress on Abscisic Acid Efflux and Compartmentation in the Roots of Two Maize Lines Differing in Drought Susceptibility

Roots of two Zea mays L. lines (drought-resistant Polj 17, and drought-susceptible F-2) were exposed to osmotic stress induced by sorbitol (osmotic potential –1.0 MPa). The following parameters were determined in cortex cells: membrane permeability for abscisic acid (ABA), ABA fluxes across membranes, pH values and ABA content in cytoplasm and vacuole. Osmotic stress induced different distribution of ABA within cell compartments in the investigated lines. ABA transport in the F-2 line occurred according to the intracellular pH gradient and the anion trap concept. In Polj 17, however, osmotic stress did not cause any significant effect on pH gradient and compartmental ABA content, but had a stimulating effect on ABA efflux from cytoplasm to apoplast and than via xylem to the leaf. These findings indicate different mechanisms of ABA transport between the investigated lines in response to osmotic stress.     

16S rDNA sequence analysis of Xylella fastidiosa strains

The 16S rDNA encoding the small subunit ribosomal RNA were amplified by PCR, cloned, and sequenced from 16 strains of Xylella fastidiosa originating from nine different hosts. In pair-wise comparisons, X. fastidiosa strains showed a maximum variation of 1.0% or 14 nucleotide positions. When all 16 sequences were considered as a set, 54 variable positions were found. Analysis of the sequence data indicated that the X. fastdiosa strains formed three rDNA groups. Group one includes Pierce's disease and mulberry leaf scorch strains; Group two, periwinkle wilt, plum leaf scald, phony peach, oak leaf scorch, and elm leaf scorch strains; and Group three, citrus variegated chlorosis and coffee leaf scorch strains. All X. fastidiosa strains exhibited significantly higher levels of sequence heterogeneity (63 to 83 nucleotide positions) when compared to species from Xanthomonas and Stenotrophomonas. Our data demonstrate that 16S rDNA sequence data could provide valuable information for future classification of X. fastidiosa at the sub-species level.     

Prevalence of Borrelia burgdorferi sensu lato genospecies in Ixodes ricinus ticks in Europe: a metaanalysis

In Europe, Borrelia burgdorferi genospecies causing Lyme borreliosis are mainly transmitted by the tick Ixodes ricinus. Since its discovery, B. burgdorferi has been the subject of many epidemiological studies to determine its prevalence and the distribution of the different genospecies in ticks. In the current study we systematically reviewed the literature on epidemiological studies of I. ricinus ticks infected with B. burgdorferi sensu lato. A total of 1,186 abstracts in English published from 1984 to 2003 were identified by a PubMed keyword search and from the compiled article references. A multistep filter process was used to select relevant articles; 110 articles from 24 countries contained data on the rates of infection of I. ricinus with Borrelia in Europe (112,579 ticks), and 44 articles from 21 countries included species-specific analyses (3,273 positive ticks). These data were used to evaluate the overall rate of infection of I. ricinus with Borrelia genospecies, regional distributions within Europe, and changes over time, as well as the influence of different detection methods on the infection rate. While the infection rate was significantly higher in adults (18.6%) than in nymphs (10.1%), no effect of detection method, tick gender, or collection period (1986 to 1993 versus 1994 to 2002) was found. The highest rates of infection of I. ricinus were found in countries in central Europe. B. afzelii and B. garinii are the most common Borrelia species, but the distribution of genospecies seems to vary in different regions in Europe. The most frequent coinfection by Borrelia species was found for B. garinii and B. valaisiana.     

Biochemical characterization of an exonuclease from Arabidopsis thaliana reveals similarities to the DNA exonuclease of the human Werner syndrome protein

The human Werner syndrome protein (hWRN-p) possessing DNA helicase and exonuclease activities is essential for genome stability. Plants have no homologue of this bifunctional protein, but surprisingly the Arabidopsis genome contains a small open reading frame (ORF) (AtWRNexo) with homology to the exonuclease domain of hWRN-p. Expression of this ORF in Escherichia coli revealed an exonuclease activity for AtWRN-exo-p with similarities but also some significant differences to hWRN-p. The protein digests recessed strands of DNA duplexes in the 3' --> 5' direction but hardly single-stranded DNA or blunt-ended duplexes. In contrast to the Werner exonuclease, AtWRNexo-p is also able to digest 3'-protruding strands. DNA with recessed 3'-PO4 and 3'-OH termini is degraded to a similar extent. AtWRNexo-p hydrolyzes the 3'-recessed strand termini of duplexes containing mismatched bases. AtWRNexo-p needs the divalent cation Mg2+ for activity, which can be replaced by Mn2+. Apurinic sites, cholesterol adducts, and oxidative DNA damage (such as 8-oxoadenine and 8-oxoguanine) inhibit or block the enzyme. Other DNA modifications, including uracil, hypoxanthine and ethenoadenine, did not inhibit AtWRNexo-p. A mutation of a conserved residue within the exonuclease domain (E135A) completely abolished the exonucleolytic activity. Our results indicate that a type of WRN-like exonuclease activity seems to be a common feature of the DNA metabolism of animals and plants.     

Structural Requirements of Abscisic Acid (ABA) and its Impact on Water Flow During Radial Transport of ABA Analogues Through Maize Roots

The effect of (+) (ABA) and (?)-abscisic acid and nine ABA metabolites, precursors or derivatives on radial water movement through maize roots, was investigated using a suction technique (Freundl and others 1998). (+)-ABA, (+)- and (?)-abscisyl aldehyde, (+)-8?-hydroxymethyl ABA, (+)-8?-methylene, and (+)-8?-acetylene ABA stimulated radial water transport. (?)-ABA, phaseic acid, and (+)-8?-acetylene methyl ABA were ineffective. ELISA analysis for ABA detected and apparent increase of free ABAxyl in xylem sap of excised root systems that were perfused with either (+)-abscisyl aldehyde, (+)-8?-methylene, (+)8?-acetylene-ABA, or ABA-glucose ester. The analogues (+)-8?-hydroxymethyl ABA and (?)-abscisyl aldehyde passed the cortex of maize roots without changing the ABAxyl. The data from this study permit conclusions about the structural requirements for hormonal regulation of hydraulic conductivity.     

Polymerase chain reaction in diagnosis of Borrelia burgdorferi infections and studies on taxonomic classification

Lyme borreliosis caused by the spirochete Borrelia burgdorferi is now the most common vectorborne disease in North America, Europe and Asia. It is a multisystemic infection which may cause skin, neurological, cardiac or rheumatologic disorders. The aims of the present thesis were: (i) to develop a PCR assay for direct detection of B. burgdorferi DNA and to evaluate the diagnostic utility of PCR in clinical specimens from patients with Lyme borreliosis and (ii) to study the taxonomic classification of B. burgdorferi isolates and its implications for epidemiology and clinical presentation. Laboratory diagnosis of Lyme borreliosis by direct demonstration of B. burgdorferi in clinical specimens would compared to current serology allow (i) optimal specificity, (ii) increased sensitivity during the first weeks of infection, when the antibody response is not yet detectable and (iii) discrimination between ongoing and past infection. Due to the extreme paucity of spirochetes in clinical specimens neither in vitro culture nor antigen detection had yielded a sufficient diagnostic sensitivity. Thus the recently introduced highly sensitive PCR methodology could be a solution and was thus studied. Assays for PCR amplification and subsequent identification of B. burgdorferi specific sequences were established and used. For all assays the analytical sensitivity was a few genome copies using purified DNA as template. The efficacy of PCR was initially evaluated using tissue samples from experimentally infected gerbils in order to start with biological samples a priori known to contain B. burgdorferi. B. burgdorferi DNA was detectable in 88% of the specimens. Thus the diagnostic sensitivity of PCR was comparable to and even higher than in vitro culture. PCR was significantly more sensitive than a histological B. burgdorferi specific immunophosphatase-staining method. The utility of the PCR was then tested for identification of B. burgdorferi DNA in skin biopsies from 31 patients with erythema migrans. The sensitivity of PCR was 71%, which was superior to culture and serology. Based on own and otherwise published results there is clear evidence for PCR being the most sensitive and specific test for detection of B. burgdorferi in skin biopsies from patients with both early and late dermatoborreliosis. However, since the clinical diagnosis of dermatoborreliosis in most instances is easy, an invasive procedure as a skin biopsy, will only be justified in patients with an atypical clinical presentation. The most frequent and serious manifestation of disseminated Lyme borreliosis is neuroborreliosis. PCR was applied to 190 patients with untreated and confirmed neuroborreliosis. B. burgdorferi DNA was detectable in 17-21% of CSF samples from patients with neuroborreliosis. In patients with very early neuroborreliosis (< 2 weeks), still being negative for specific intrathecal antibody synthesis, a positive PCR was more frequent than in patients with longer disease duration. PCR can be used as a diagnostic aid in these patients. However, in general the measurement of specific intrathecal antibody production in patients with neuroborreliosis was superior to PCR. In urine samples from patients with Lyme borreliosis the diagnostic sensitivity varied, generally showing a low reproducibility. Urine is thus not regarded as a suitable sample source for B. burgdorferi PCR. The reason may be the variable presence of Taq polymerase inhibitors. Based on a semi-quantitative detection system for amplicons, reflecting the input amount of specific DNA and thus the density of spirochetes in the clinical samples high amounts of DNA were found in skin biopsies whereas especially in urine the amount of DNA was low. When the present study was initiated there was no accepted classification of B. burgdorferi. A heterogeneity among B. burgdorferi strains might have important implications for understanding the epidemiology and different clinical presentations (dermatoborreliosis versus neuroborreliosis) and courses (self-limiting versus chronic disease). Furthermore, strain differences were of importance for selection of suitable antigens for diagnostic assays and for vaccine development. Since then, B. burgdorferi isolates have been studied by phenotypic and genotypic traits and have been shown to be highly heterogeneous. Our first approach was to genotype a panel of human B. burgdorferi isolates by restriction fragment length polymorphism (RFLP) of three genes. Thereafter, sequencing and dideoxy fingerprinting of ospA was applied. By RFLP the strains could be differentiated into two to five groups. The RFLP classification was compared with four different phenotypic and genotypic methods including the rRNA typing. Results obtained with the different methods correlated highly and confirmed the meanwhile accepted taxonomic classification by Baranton et al., According to this the term B. burgdorferi sensu lato comprises three different human pathogenic genospecies B. burgdorferi sensu stricto, B. garinii and B. afzelii. All three genospecies have been isolated among Danish patients with Lyme borreliosis and are thus prevalent in Denmark. Since isolation of B. burgdorferi from patients with Lyme borreliosis is laborious and often unsuccessful molecular typing methods based on PCR are recommended obviating the need for isolation by prior culture. Of special interest was to study a possible association of neuroborreliosis to certain B. burgdorferi genospecies, indicating species depended organotropism. By RFLP all six CSF isolates tested belonged to B. garinii and that 6 out of 7 isolates from patients with acrodermatitis chronica atrophicans belonged to B. afzelii. Due to the low culture yield of B. burgdorferi from CSF, the association of B. garinii and neuroborreliosis was further studied by sequence analysis and dideoxyfingerprinting analysis of ospA PCR amplicons obtained from CSF samples from patients with neuroborreliosis. Phylogenetic analysis showed that in 11 out of 13 patients B. garinii DNA was found in CSF. These data strongly supports the hypothesis that B. garinii is the principal agent of Lyme neuroborreliosis in Europe. Similarly it was shown that B. afzelii is associated with acrodermatitis chronica atrophicans and thus dermatoborreliosis. Due to a strain dependent different selection pressure in culture only PCR based methods can be used to answer whether mixed infection in patients specimens occur. Our data indicate that mixed infections in humans if ever are rare.     

Nucleolus,nucleolar chromosomes,and nucleolus-associated chromatin from early diplotene to dictyotene in the human oocyte

Summary The shape, relationships, relative DNA content, and nucleolar activity of the short arm of acrocentric bivalents were studied in human oocytes from early diplotene to dictyotene. At the beginning of diplotene, the short arms of the previously paired chromosomes were again separated and displayed the same morphological features as in mitotic prophase chromosomes. They were connected only with the nucleolus. In situ hybridization and silver staining showed that the nucleolar organizer regions (NORs) were located in the peripheral region of the nucleolus. Tritiated-uridine incorporation was active. At birth, the relationships of the acrocentric short arms showed increasing complexity. The chromosomes ended in nucleolus-associated chromatin blocks of irregular shape, containing large quantities of DNA as demonstrated by intense binding of³H-actinomycin D. The number of chromosomes converging on these chromatin blocks exceeded the number of acrocentrics, suggesting that heterochromatic regions of other chromosomes were associated with the short arm of acrocentrics. In the electron microscope, the NORs were represented by fibrillar centers located on the periphery of the nucleolus and consistently connected with the blocks of dense chromatin. These relationships remained unchanged in the primordial oocyte in the adult ovary. Persistence of³H-uridine uptake showed that the oocyte was not at a resting stage. The possible cytogenetic consequences of these observations are discussed.     

Fluctuations of the Ca2+-activated K+ current in Aplysia neurones

Fluctuations of the Ca2+-activated K+ current were measured in identified Aplysia neurones under voltage clamp conditions. The amplitude of IK,Ca was manipulated by ionophoretic injections of Ca2+. At small amplitudes of Ca2+-activated outward currents the variance of the Ca2+-activated current fluctuations increases linearly with the mean outward current. The single-channel conductance estimated from the variance of the fluctuations and the mean outward current is 11 +/- 3 pS at -30 mV. Power spectra of the Ca2+-activated K+ current can be fitted by the sum of two Lorentzian components with corner frequencies of about 10 Hz and 120 Hz.