Amyloplasts are necessary for full gravitropic sensitivity in roots of Arabidopsis thaliana

The observation that a starchless mutant (TC7) of Arabidopsis thaliana (L.) Heynh. is gravitropic (T. Caspar and B.G. Pickard, 1989, Planta 177, 185–197) raises questions about the hypothesis that starch and amyloplasts play a role in gravity perception. We compared the kinetics of gravitropism in this starchless mutant and the wild-type (WT). Wild-type roots are more responsive to gravity than TC7 roots as judged by several parameters: (1) Vertically grown TC7 roots were not as oriented with respect to the gravity vector as WT roots. (2) In the time course of curvature after gravistimulation, curvature in TC7 roots was delayed and reduced compared to WT roots. (3) TC7 roots curved less than WT roots following a single, short (induction) period of gravistimulation, and WT, but not TC7, roots curved in response to a 1-min period of horizontal exposure. (4) Wild-type roots curved much more than TC7 roots in response to intermittent stimulation (repeated short periods of horizontal exposure); WT roots curved in response to 10 s of stimulation or less, but TC7 roots required 2 min of stimulation to produce a curvature. The growth rates were equal for both genotypes. We conclude that WT roots are more sensitive to gravity than TC7 roots. Starch is not required for gravity perception in TC7 roots, but is necessary for full sensitivity; thus it is likely that amyloplasts function as statoliths in WT Arabidopsis roots. Furthermore, since centrifugation studies using low gravitational forces indicated that starchless plastids are relatively dense and are the most movable component in TC7 columella cells, the starchless plastids may also function as statoliths.Abbreviations S2 story two - S3 story three - WT wild-type     

Periplasmic binding protein structure and function. Refined X-ray structures of the leucine/isoleucine/valine-binding protein and its complex with leucine

The three-dimensional structure of the native unliganded form of the Leu/Ile/Val-binding protein (Mr = 36,700), an essential component of the high-affinity active transport system for the branched aliphatic amino acids in Escherichia coli, has been determined and further refined to a crystallographic R-factor of 0.17 at 2.4 A resolution. The entire structure consists of 2710 non-hydrogen atoms from the complete sequence of 344 residues and 121 ordered water molecules. Bond lengths and angle distances in the refined model have root-mean-square deviations from ideal values of 0.05 A and 0.10 A, respectively. The overall shape of the protein is a prolate ellipsoid with dimensions of 35 A x 40 A x 70 A. The protein consists of two distinct globular domains linked by three short peptide segments which, though widely separated in the sequence, are proximal in the tertiary structure and form the base of the deep cleft between the two domains. Although each domain is built from polypeptide segments located in both the amino (N) and the carboxy (C) terminal halves, both domains exhibit very similar supersecondary structures, consisting of a central beta-sheet of seven strands flanked on either side by two or three helices. The two domains are far apart from each other, leaving the cleft wide open by about 18 A. The cleft has a depth of about 15 A and a base of about 14 A x 16 A. Refining independently the structure of native Leu/Ile/Val-binding protein crystals soaked in a solution containing L-leucine at 2.8 A resolution (R-factor = 0.15), we have been able to locate and characterize an initial, major portion of the substrate-binding site of the Leu/Ile/Val-binding protein. The binding of the L-leucine substrate does not alter the native crystal structure, and the L-leucine is lodged in a crevice on the wall of the N-domain, which is in the inter-domain cleft. The L-leucine is held in place primarily by hydrogen-bonding of its alpha-ammonium and alpha-carboxylate groups with main-chain peptide units and hydroxyl side-chain groups; there are no salt-linkages. The charges on the leucine zwitterion are stabilized by hydrogen-bond dipoles. The side-chain of the L-leucine substrate lies in a depression lined with non-polar residues, including Leu77, which confers specificity to the site by stacking with the side-chain of the leucine substrate.(ABSTRACT TRUNCATED AT 400 WORDS)     

Macromolecular differentiation of Golgi stacks in root tips ofArabidopsis andNicotiana seedlings as visualized in high pressure frozen and freeze-substituted samples

Summary The plant root tip represents a fascinating model system for studying changes in Golgi stack architecture associated with the developmental progression of meristematic cells to gravity sensing columella cells, and finally to young and old, polysaccharideslime secreting peripheral cells. To this end we have used high pressure freezing in conjunction with freeze-substitution techniques to follow developmental changes in the macromolecular organization of Golgi stacks in root tips ofArabidopsis andNicotiana. Due to the much improved structural preservation of all cells under investigation, our electron micrographs reveal both several novel structural features common to all Golgi stacks, as well as characteristic differences in morphology between Golgi stacks of different cell types.Common to all Golgi stacks are clear and discrete differences in staining patterns and width of cis, medial and trans cisternae. Cis cisternae have the widest lumina (30 nm) and are the least stained. Medial cisternae are narrower (20 nm) and filled with more darkly staining products. Most trans cisternae possess a completely collapsed lumen in their central domain, giving rise to a 4–6 nm wide dark line in cross-sectional views. Numerous vesicles associated with the cisternal margins carry a non-clathrin type of coat. A trans Golgi network with clathrin coated vesicles is associated with all Golgi stacks except those of old peripheral cells. It is easily distinguished from trans cisternae by its blebbing morphology and staining pattern. The zone of ribosome exclusion includes both the Golgi stack and the trans Golgi network.Intercisternal elements are located exclusively between trans cisternae of columella and peripheral cells, but not meristematic cells. In older peripheral cells only trans cisternae exhibit slime-related staining. Golgi stacks possessing intercisternal elements also contain parallel rows of freeze-fracture particles in their trans cisternal membranes. We propose that intercisternal elements serve as anchors of enzyme complexes involved in the synthesis of polysaccharide slime molecules to prevent the complexes from being dragged into the forming secretory vesicles by the very large slime molecules. In addition, we draw attention to the similarities in composition and apparent site of synthesis of xyloglucans and slime molecules.Dedicated to the memory of Professor Oswald Kiermayer     

The ecosystem wilting point defines drought response and recovery of a Quercus-Carya forest

Soil and atmospheric droughts increasingly threaten plant survival and productivity around the world. Yet, conceptual gaps constrain our ability to predict ecosystem-scale drought impacts under climate change. Here, we introduce the ecosystem wilting point (Ψ_EWP), a property that integrates the drought response of an ecosystem's plant community across the soil–plant–atmosphere continuum. Specifically, Ψ_EWP defines a threshold below which the capacity of the root system to extract soil water and the ability of the leaves to maintain stomatal function are strongly diminished. We combined ecosystem flux and leaf water potential measurements to derive the Ψ_EWP of a Quercus-Carya forest from an “ecosystem pressure–volume (PV) curve,” which is analogous to the tissue-level technique. When community predawn leaf water potential (Ψ_pd) was above Ψ_EWP (=−2.0 MPa), the forest was highly responsive to environmental dynamics. When Ψ_pd fell below Ψ_EWP, the forest became insensitive to environmental variation and was a net source of carbon dioxide for nearly 2 months. Thus, Ψ_EWP is a threshold defining marked shifts in ecosystem functional state. Though there was rainfall-induced recovery of ecosystem gas exchange following soaking rains, a legacy of structural and physiological damage inhibited canopy photosynthetic capacity. Although over 16 growing seasons, only 10% of Ψ_pd observations fell below Ψ_EWP, the forest is commonly only 2–4 weeks of intense drought away from reaching Ψ_EWP, and thus highly reliant on frequent rainfall to replenish the soil water supply. We propose, based on a bottom-up analysis of root density profiles and soil moisture characteristic curves, that soil water acquisition capacity is the major determinant of Ψ_EWP, and species in an ecosystem require compatible leaf-level traits such as turgor loss point so that leaf wilting is coordinated with the inability to extract further water from the soil.     

Detection of Vibrio cholerae O1 in the aquatic environment by fluorescent-monoclonal antibody and culture methods.

Vibrio cholerae O1 in plankton samples collected from ponds and rivers between February 1987 and January 1990 in Matlab, Bangladesh, was detected by the fluorescent-monoclonal antibody (FA) technique. Samples were collected at sites which were monitored fortnightly (fixed sites) as well as at sites that were part of a case-control study. FA results were compared with those obtained by conventional culture methods (CM). A total of 876 samples were collected; V. cholerae O1 was detected in 563 samples (64.27%) by the FA method and in 3 samples (0.34%) by CM. Of the fixed-site plankton samples, 439 (63.62%) were positive by FA and none were positive by CM. Of the 93 case sites sampled on the day after the occurrence of a case of cholera, 73 (78.49%) were positive for V. cholerae O1 by FA and 3 (3.2%) were positive by CM. In comparison, of the 93 first-day sample collections at control sites at the time a case of cholera occurred, only 51 (54.83%) were positive by FA and none were positive by CM. From the data, it is concluded that V. cholerae O1 is present throughout the year in the ponds and rivers of Bangladesh that were examined in this study and that V. cholerae can be detected by FA but not always by CM. The FA procedure was found to be very useful in detecting V. cholerae in plankton, with which it was associated and often occurred in large numbers in the nonculturable stage. Thus, studies investigating the significance of the role of environmental factors in the epidemiology of cholera can be performed effectively by using FA. Such studies are in progress.     

Pandemic strains of O3:K6 Vibrio parahaemolyticus in the aquatic environment of Bangladesh

A total of 1500 environmental strains of Vibrio parahaemolyticus, isolated from the aquatic environment of Bangladesh, were screened for the presence of a major V. parahaemolyticus virulence factor, the thermostable direct haemolysin (tdh) gene, by the colony blot hybridization method using a digoxigenin-labeled tdh gene probe. Of 1500 strains, 5 carried the tdh sequence, which was further confirmed by PCR using primers specific for the tdh gene. Examination by PCR confirmed that the 5 strains were V. parahaemolyticus and lacked the thermostable direct haemolysin-related haemolysin (trh) gene, the alternative major virulence gene known to be absent in pandemic strains. All 5 strains gave positive Kanagawa phenomenon reaction with characteristic beta-haemolysis on Wagatsuma agar medium. Southern blot analysis of the HindIII-digested chromosomal DNA demonstrated, in all 5 strains, the presence of 2 tdh genes common to strains positive for Kanagawa phenomenon. However, the 5 strains were found to belong to 3 different serotypes (O3:K29, O4:K37, and O3:K6). The 2 with pandemic serotype O3:K6 gave positive results in group-specific PCR and ORF8 PCR assays, characteristics unique to the pandemic clone. Clonal variations among the 5 isolates were analyzed by comparing RAPD and ribotyping patterns. Results showed different patterns for the 3 serotypes, but the pattern was identical among the O3:K6 strains. This is the first report on the isolation of pandemic O3:K6 strains of V. parahaemolyticus from the aquatic environment of Bangladesh.     

Pre-Stimulus Sham TMS Facilitates Target Detection

Transcranial magnetic stimulation (TMS) allows non-invasive manipulation of brain activity during active task performance. Because every TMS pulse is accompanied by non-neural effects such as a clicking sound and somato-sensation on the head, control conditions are required to ensure that changes in task behavior are indeed due to the induced neural effects. However, the non-neural effects of TMS in the context of a given task performance are largely unknown and, consequently, it is unclear what constitutes a valid control condition. We explored the non-neural effects of TMS on visual target detection. Participants received single pulse sham TMS to each hemisphere at different time points prior to target appearance during a visual target detection task. It was hypothesized that the clicking sound of a sham TMS pulse differentially affects performance depending on the location of the coil and the timing of the pulse.Our results show that, first, sham TMS caused a facilitation of reaction times when preceding the target stimulus by 150, 200, and 250 ms, whereas earlier and later time windows were not effective. Second, positioning the TMS coil ipsilateral instead of contralateral relative to the target stimulus improved reaction times. Third, infrequent noTMS trials that were interleaved with sham TMS trials had oddball-like properties resulting in increased reaction times during noTMS. The clicking sound produced by sham TMS influences task performance in multiple ways. These non-neural effects of TMS need to be controlled for in TMS research and the present findings provide an empirical basis for deciding what constitutes a valid control condition.     

Mediation of Clathrin-Dependent Trafficking during Cytokinesis and Cell Expansion by Arabidopsis STOMATAL CYTOKINESIS DEFECTIVE Proteins

STOMATAL CYTOKINESIS DEFECTIVE1 (SCD1) encodes a putative Rab guanine nucleotide exchange factor that functions in membrane trafficking and is required for cytokinesis and cell expansion in Arabidopsis thaliana. Here, we show that the loss of SCD2 function disrupts cytokinesis and cell expansion and impairs fertility, phenotypes similar to those observed for scd1 mutants. Genetic and biochemical analyses showed that SCD1 function is dependent upon SCD2 and that together these proteins are required for plasma membrane internalization. Further specifying the role of these proteins in membrane trafficking, SCD1 and SCD2 proteins were found to be associated with isolated clathrin-coated vesicles and to colocalize with clathrin light chain at putative sites of endocytosis at the plasma membrane. Together, these data suggest that SCD1 and SCD2 function in clathrin-mediated membrane transport, including plasma membrane endocytosis, required for cytokinesis and cell expansion.     

Tuberculous Lymphadenitis in Northern Ethiopia: In a Public Health and Microbiological Perspectives

Background

The actual burden and causative agent of tuberculous lymphadenitis (TBLN) cases is not well known due to lack of strong surveillance system and diagnostic facilities in Ethiopia. This study was conducted to determine the prevalence of TBLN, its causative agent and risk factors for acquiring this infection.

Methods

A cross-sectional study was conducted from April to May 2012 at four main hospitals and one diagnostic clinic located in northern Ethiopia. Fine needle aspirates (FNAs) from TBLN suspects were taken for acid fast bacilli (AFB) microscopy, culture and molecular typing.

Results

Among 437 aspirates, culture yielded AFB in 226 (51.7%) of cases. Sixty one culture negative cases (30.5% of 200 cases) were positive by Xpert MTB/RIF test. Moreover, a rifampicin resistant AFB was detected from culture negative cases. The overall prevalence of FNAs positive TBLN cases was 65.8 %. The BacT/AlerT 3D system proved to be a more rapid method with higher recovery rate than Lowenstein-Jensen (L-J) and/or Gottsacker media (P<0.0001). Molecular typing identified all culture positive isolates as M.tuberculosis. The main risk factors for TBLN were pediatric age (OR 2.8, 95% CI, 1.09- 7.05) and cough (OR 2, 95%CI, 1.09-3.7).

Conclusions

The results of this study revealed a high prevalence of TBLN in the study sites and that pediatric age and cough are key predictors of the disease. TBLN is an important public health problem that needs to be addressed in the area. It is important to note that MDR strains of TB could be involved and aetiological confirmation and drug sensitivity testing of TBLN isolates should be expanded. Further studies on the M.tuberculosis lineages, circulating strains and transmission dynamics, are recommended.     

Quantitative Bioluminescent Imaging of Pre-Erythrocytic Malaria Parasite Infection Using Luciferase-Expressing Plasmodium yoelii

The liver stages of Plasmodium parasites are important targets for the development of anti-malarial vaccine candidates and chemoprophylaxis approaches that aim to prevent clinical infection. Analyzing the impact of interventions on liver stages in the murine malaria model system Plasmodium yoelii has been cumbersome and requires terminal procedures. In vivo imaging of bioluminescent parasites has previously been shown to be an effective and non-invasive alternative to monitoring liver stage burden in the Plasmodium berghei model. Here we report the generation and characterization of a transgenic P. yoelii parasite expressing the reporter protein luciferase throughout the parasite life cycle. In vivo bioluminescent imaging of these parasites allows for quantitative analysis of P. yoelii liver stage burden and parasite development, which is comparable to quantitative RT-PCR analysis of liver infection. Using this system, we show that both BALB/cJ and C57BL/6 mice show comparable susceptibility to P. yoelii infection with sporozoites and that bioluminescent imaging can be used to monitor protective efficacy of attenuated parasite immunizations. Thus, this rapid, simple and noninvasive method for monitoring P. yoelii infection in the liver provides an efficient system to screen and evaluate the effects of anti-malarial interventions in vivo and in real-time.