N-glucosylation of cytokinins by glycosyltransferases of Arabidopsis thaliana

Cytokinins are plant hormones that can be glucosylated to form O-glucosides and N-glucosides. The glycoconjugates are inactive and are thought to play a role in homeostasis of the hormones. Although O-glucosyltransferases have been identified that recognize cytokinins, the enzymes involved in N-glucosylation have not been identified even though the process has been recognized for many years. This study utilizes a screening strategy in which 105 recombinant glycosyltransferases (UGTs) of Arabidopsis have been analyzed for catalytic activity toward the classical cytokinins: trans-zeatin, dihydrozeatin, N(6)-benzyladenine, N(6)-isopentenyladenine, and kinetin. Five UGTs were identified in the screen. UGT76C1 and UGT76C2 recognized all cytokinins and glucosylated the hormones at the N(7) and N(9) positions. UGT85A1, UGT73C5, and UGT73C1 recognized trans-zeatin and dihydrozeatin, which have an available hydroxyl group for glucosylation and formed the O-glucosides. The biochemical characteristics of the N-glucosyltransferases were analyzed, and highly effective inhibitors of their activities were identified. Constitutive overexpression of UGT76C1 in transgenic Arabidopsis confirmed that the recombinant enzyme functioned in vivo to glucosylate cytokinin applied to the plant. The role of the N-glucosyltransferases in cytokinin metabolism is discussed.     

Stimulus secretion coupling in vitro: a rapid perfusion apparatus for monitoring efflux of transmitter substances from tissue samples.

An apparatus for monitoring efflux rates of specific substances from cellular preparations is described. Tissue samples (homogenates, subcellular fractions, small tissue slices, cell suspensions etc.) are placed on a filter, perfused with several different media sequentially and aliquots of the perfusate collected at intervals of 5 sec. Under maximum perfusion rates, the changeover in perfusion media is completed in less than 1 sec, produces no detectable disturbance of the sample and allows only minimal mixing of the different media. The apparatus has been used successfully to study stimulus secretion coupling during release of the neurotransmitter [¹⁴C]γ-aminobutyric acid from synaptosomes.     

Mutations in a BTB-Kelch Protein, KLHL7, Cause Autosomal-Dominant Retinitis Pigmentosa

Retinitis pigmentosa (RP) refers to a genetically heterogeneous group of progressive neurodegenerative diseases that result in dysfunction and/or death of rod and cone photoreceptors in the retina. So far, 18 genes have been identified for autosomal-dominant (ad) RP. Here, we describe an adRP locus (RP42) at chromosome 7p15 through linkage analysis in a six-generation Scandinavian family and identify a disease-causing mutation, c.449G→A (p.S150N), in exon 6 of the KLHL7 gene. Mutation screening of KLHL7 in 502 retinopathy probands has revealed three different missense mutations in six independent families. KLHL7 is widely expressed, including expression in rod photoreceptors, and encodes a 75 kDa protein of the BTB-Kelch subfamily within the BTB superfamily. BTB-Kelch proteins have been implicated in ubiquitination through Cullin E3 ligases. Notably, all three putative disease-causing KLHL7 mutations are within a conserved BACK domain; homology modeling suggests that mutant amino acid side chains can potentially fill the cleft between two helices, thereby affecting the ubiquitination complexes. Mutations in an identical region of another BTB-Kelch protein, gigaxonin, have previously been associated with giant axonal neuropathy. Our studies suggest an additional role of the ubiquitin-proteasome protein-degradation pathway in maintaining neuronal health and in disease.     

A whole-genome single nucleotide polymorphism-based approach to trace and identify outbreaks linked to a common Salmonella enterica subsp. enterica serovar Montevideo pulsed-field gel electrophoresis type

In this study, we report a whole-genome single nucleotide polymorphism (SNP)-based evolutionary approach to study the epidemiology of a multistate outbreak of Salmonella enterica subsp. enterica serovar Montevideo. This outbreak included 272 cases that occurred in 44 states between July 2009 and April 2010. A case-control study linked the consumption of salami made with contaminated black and red pepper to the outbreak. We sequenced, on the SOLiD System, 47 isolates with XbaI PFGE pattern JIXX01.0011, a common pulsed-field gel electrophoresis (PFGE) pattern associated with isolates from the outbreak. These isolates represented 20 isolates collected from human sources during the period of the outbreak and 27 control isolates collected from human, food, animal, and environmental sources before the outbreak. Based on 253 high-confidence SNPs, we were able to reconstruct a tip-dated molecular clock phylogeny of the isolates and to assign four human isolates to the actual outbreak. We developed an SNP typing assay to rapidly discriminate between outbreak-related cases and non-outbreak-related cases and tested this assay on an extended panel of 112 isolates. These results suggest that only a very small percentage of the human isolates with the outbreak PFGE pattern and obtained during the outbreak period could be attributed to the actual pepper-related outbreak (20%), while the majority (80%) of the putative cases represented background cases. This study demonstrates that next-generation-based SNP typing provides the resolution and accuracy needed for outbreak investigations of food-borne pathogens that cannot be distinguished by currently used subtyping methods.     

Rip stop in marine algae: Minimizing the consequences of herbivore damage

Summary Structural features of marine macrophytes are generally believed to act as defences against herbivores by reducing the ability of herbivores to consume the plants. Thallus form and calcification in particular have been considered structural defences that act by reducing the probability of consumption of tissue by herbivores. Studies directly measuring the mechanical resistance of a variety of marine algae (tropical and temperate) to herbivores of two important feeding types, rasping herbivores (docoglossan limpets) and a biting herbivore (an herbivorous crab), do not support this hypothesis. I suggest that thallus form and calcification may play a more important role in minimizing the impact of herbivores by reducing the probability of subsequent tissue loss due to herbivore-induced damage. For some algal species, tissue lost subsequent to herbivore damage may greatly exceed loss due to direct consumption by herbivores. I suggest that calcification and thallus properties resulting in preferential tear directions reduce the probability of tissue loss subsequent to herbivore damage rather than prevent herbivores from removing tissue as has been suggested in the past.     

Plastic inducible morphologies are not always adaptive: The importance of time delays in a stochastic environment

Summary We present a mathematical model for predicting the expected fitness of phenotypically plastic organisms experiencing a variable environment. We assume that individuals experience two discrete environments probabilistically in time (as a Markov process) and that there are two different phenotypic states, each yielding the highest fitness in one of the two environments. We compare the expected fitness of a phenotypically fixed individual to that of an individual whose phenotype is induced to produce the better phenotype in each environment with a time lag between experiencing a new environment and realization of the new phenotype. Such time lags are common in organisms where phenotypically plastic, inducible traits have been documented. We find that although plasticity is generally adaptive when time lags are short (relative to the time scale of environmental variability), plasticity can be disadvantageous for longer lag times. Asymmetries in environmental change probabilities and/or the relative fitnesses of each phenotype strongly influence whether plasticity is favoured. In contrast to other models, our model does not require costs for plasticity to be disadvantageous; costs affect the results quantitatively, not qualitatively.     

Epidemiological Tracing of Pseudomonas aeruginosa: Antibiogram and Serotyping

The spread of a particular strain of Pseudomonas aeruginosa through a pediatric burn unit was monitored using serological typing and antibiotic susceptibility data.     

Immunocytochemical localization of polyamines during attachment and spreading of retinal pigment epithelial and intestinal epithelial cells

In order to form and maintain a protective barrier for photoreceptors, the retinal pigment epithelium relies on integrin signaling and related pathways to form adhesion complexes, undergo cell spreading, and establish a confluent cellular monolayer. Polyamines are multifunctional polycations that are essential for cell attachment and spreading, although their exact mechanisms of action are as yet unclear. We report new immunocytochemical evidence suggesting that in the cells of retinal pigment epithelium and also the intestinal epithelium, polyamines are present in a population of intracellular vesicles that appear transiently during initial stages of cell spreading. In newly attached cells with minimal spreading, the vesicles are seen near the nucleus, whereas in more highly spread cells, the vesicles are localized to the plasma membrane, near, but not precisely co-localized with an enzyme marker for adhesion complexes, focal adhesion kinase. We also observe pronounced nuclear staining in newly attached cells that have not spread, whereas this staining is decreased in cells that have spread. Nuclear staining has been previously reported in other cell types and has been attributed to DNA binding of polyamines, which is known to stabilize chromatin structure. We hypothesize that the appearance of polyamine vesicles near focal adhesions of cells undergoing attachment and spreading may reflect the mechanism by which polyamine pools are targeted to appropriate interaction sites necessary for the assembly of adhesion complexes. Alternatively, the vesicles could represent the mechanism by which polyamines are removed from the nucleus and possibly released from the cell.