Desmoplakin Is Required Early in Development for Assembly of Desmosomes and Cytoskeletal Linkage

Desmosomes first assemble in the E3.5 mouse trophectoderm, concomitant with establishment of epithelial polarity and appearance of a blastocoel cavity. Throughout development, they increase in size and number and are especially abundant in epidermis and heart muscle. Desmosomes mediate cell–cell adhesion through desmosomal cadherins, which differ from classical cadherins in their attachments to intermediate filaments (IFs), rather than actin filaments. Of the proteins implicated in making this IF connection, only desmoplakin (DP) is both exclusive to and ubiquitous among desmosomes. To explore its function and importance to tissue integrity, we ablated the desmoplakin gene. Homozygous −/− mutant embryos proceeded through implantation, but did not survive beyond E6.5. Mutant embryos proceeded through implantation, but did not survive beyond E6.5. Surprisingly, analysis of these embryos revealed a critical role for desmoplakin not only in anchoring IFs to desmosomes, but also in desmosome assembly and/or stabilization. This finding not only unveiled a new function for desmoplakin, but also provided the first opportunity to explore desmosome function during embryogenesis. While a blastocoel cavity formed and epithelial cell polarity was at least partially established in the DP (−/−) embryos, the paucity of desmosomal cell–cell junctions severely affected the modeling of tissue architecture and shaping of the early embryo.     

Isolation of Rhodospirillum centenum Mutants Defective in Phototactic Colony Motility by Transposon Mutagenesis

The purple photosynthetic bacterium Rhodospirillum centenum is capable of forming swarm colonies that rapidly migrate toward or away from light, depending on the wavelength of excitation. To identify components specific for photoperception, we conducted mini-Tn5-mediated mutagenesis and screened approximately 23,000 transposition events for mutants that failed to respond to either continuous illumination or to a step down in light intensity. A majority of the ca. 250 mutants identified lost the ability to form motile swarm cells on an agar surface. These cells appeared to contain defects in the synthesis or assembly of surface-induced lateral flagella. Another large fraction of mutants that were unresponsive to light were shown to be defective in the formation of a functional photosynthetic apparatus. Several photosensory mutants also were obtained with defects in the perception and transmission of light signals. Twelve mutants in this class were shown to contain disruptions in a chemotaxis operon, and five mutants contained disruptions of components unique to photoperception. It was shown that screening for photosensory defective R. centenum swarm colonies is an effective method for genetic dissection of the mechanism of light sensing in eubacteria.Behavioral change in response to alterations in the quality and quantity of light in the environment is a ubiquitous trait among motile photosynthetic bacteria. Three distinct types of responses to light have been described in the literature (14, 19, 36, 37). The scotophobic response (fear of darkness) is characterized by a tumbling, stop, or reversal that occurs when a swimming bacterium experiences a temporal, or spatial, step down in light intensity. Photokinesis describes an alteration in the rate of motility caused by differences in light intensity. A phototactic response, which has been studied most extensively in algae and cyanobacteria, involves an oriented movement of a cell toward or away from a light source (19). An important distinction is that the direction of irradiation is not relevant to scotophobic or photokinetic responses, whereas it is a critical determinant in phototaxis. Thus, phototactic organisms are uniquely capable of migrating towards a light source, irrespective of whether they are going up or down a gradient of light intensity (37).The various photosensory behaviors exhibited by anoxygenic photosynthetic bacteria have been studied mainly by physiological and biochemical tests, with little supporting genetic data (3, 4, 8, 9, 13, 16, 27, 38). The few genetic tests that have been undertaken have demonstrated that mutations which functionally impair the photosystem also disrupt the ability of cells to respond to light (3, 20). This indicates that a product of photosynthesis, such as the generation of proton motive force or photosynthesis-driven electron transfer, is most likely the signal that controls photosensory behavior, rather than direct absorption of light by a chromophore-containing receptor. This conclusion is supported by recent physiological studies which have shown that specific inhibitors of cyclic photosynthesis-driven electron transport inhibit photosensory behavior in Rhodobacter sphaeroides (13, 16) and Rhodospirillum centenum (38). By using a site-directed mutational approach, we have shown that the scotophobic and phototactic responses of the purple nonsulfur photosynthetic bacterium R. centenum involve components of the chemotaxis phosphorylation cascade (25, 26). This suggests that a sensor of photosynthetic activity may have features similar to that of chemoreceptors. However, which component of the photosynthesis electron transfer chain is being sensed and what is actually sensing alterations in electron transfer are unknown.To identify components responsible for prokaryotic behavioral responses to light, it is essential that techniques be developed for the isolation of mutants that are specifically defective in photosensory behavior. One of the reasons why screens for photosensory mutants have not been developed is the inherent difficulty of assaying for photosensory behavior. Until recently, screening for such mutants involved the onerous task of microscopically assaying individual cells from liquid-grown cultures for a response to a step up or down in light intensity. Since statistically meaningful results require that multiple cells be assayed, this “brute force” approach is infeasible. A significant advance in the isolation of prokaryotic photosensory mutants was recently provided by our observation that colonies of the purple photosynthetic bacterium R. centenum are capable of macroscopic phototactic motility (36, 37). Cells of R. centenum are dimorphic, existing in liquid medium as swim cells bearing a single polar flagellum or as hyperflagellated swarm cells on solid surfaces (36, 37). A unique feature of R. centenum swarming colonies is that they are capable of migrating rapidly (up to 75 mm/h) toward an infrared light source or away from a visible light source (36, 37). This behavior allows us to rapidly screen for mutants that are deficient in photosensory responses by simply assaying colonies for aberrant light-directed migration. In this study, we have utilized mini-Tn5-mediated mutagenesis to isolate numerous mutants that exhibit defects in light-directed motility. The phenotypes of specific classes of mutants provide some unique observations on photosensory behavior, as well as on the mechanism of swim cell to swarm cell differentiation.     

Quantification of the antipsychotics flupentixol and haloperidol in human serum by high-performance liquid chromatography with ultraviolet detection

A high-performance liquid chromatography (HPLC) method was developed for quantification of both isomers of the thioxanthene neuroleptic flupentixol and of the butyrophenone derivative haloperidol in human serum. After extraction with diethyl ether–n-heptane (50:50, v/v), an isocratic normal-phase HPLC system with a Hypersil cyanopropyl silica column (250×4.6 mm, 5 μm particle size) was used with ultraviolet detection at 254 nm and elution with a mixture of 920 ml acetonitrile, 110 ml methanol, 30 ml 0.1 M ammonium acetate, and 50 μl triethylamine. The limit of quantitation of 0.5 ng/ml and 0.3 ng/ml for flupentixol and haloperidol, respectively, was sufficient to quantify both compounds in serum after administration of clinically adjusted doses. The suitability of the described method for therapeutic drug monitoring and clinical pharmacokinetic studies was assessed by analysis of more than 100 trough level serum samples.     

Rapid and efficient transformation of diploid Medicago truncatula and Medicago sativa ssp. falcata lines improved in somatic embryogenesis

We describe a simple and efficient protocol for regeneration-transformation of two diploid Medicago lines: the annual M. truncatula R108-1(c3) and the perennial M. sativa ssp. falcata (L.) Arcangeli PI.564263 selected previously as highly embryogenic genotypes. Here, embryo regeneration of R108-1 to complete plants was further improved by three successive in vitro regeneration cycles resulting in the line R108-1(c3). Agrobacterium tumefaciens-mediated transformation of leaf explants was carried out with promoter-gus constructs of two early nodulins (MsEnod12A and MsEnod12B) and one late nodulin (Srglb3). The transgenic plants thus produced on all explants within 3–4 months remained diploid and were fertile. This protocol appears to be the most efficient and fastest reported so far for leguminous plants. Received: 18 March 1997 / Revision received: 25 June 1997 / Accepted: 15 July 1997     

Application of Kohonen's self-organizing feature map algorithm to cortical maps of orientation and direction preference

Cortical maps of orientation preference in cats, ferrets and monkeys contain numerous half-rotation point singularities. Experimental data have shown that direction preference also has a smooth representation in these maps, with preferences being for the most part orthogonal to the axis of preferred orientation. As a result, the orientation singularities induce an extensive set of linear fractures in the direction map. These fractures run between and connect nearby point orientation singularities. Their existence appears to pose a puzzle for theories that postulate that cortical maps maximize continuity of representation, because the fractures could be avoided if the orientation map contained full-rotation singularities. Here we show that a dimension-reduction model of cortical map formation, which implements principles of continuity and completeness, produces an arrangement of linear direction fractures connecting point orientation singularities which is similar to that observed experimentally. We analyse the behaviour of this model and suggest reasons why the model maps contain half-rotation rather than full-rotation orientation singularities.     

Characterization of a light-responding trans-activator responsible for differentially controlling reaction center and light-harvesting-I gene expression in Rhodobacter capsulatus.

The purple nonsulfur photosynthetic bacterium Rhodobacter capsulatus regulates synthesis of its photosystem in response to two environmental stimuli, oxygen tension and light intensity. Here we describe the identification and characterization of the trans-acting regulatory gene hvrA, which we show is involved in differentially controlling reaction center and light-harvesting gene expression in response to alterations in light intensity. An hvrA mutant strain is shown to lack the capability to trans-activate light-harvesting-I and reaction center gene expression but retain normal light-harvesting-II and photopigment regulation, in response to a reduction in light intensity. As a consequence of altered expression, hvrA mutant strains exhibit reduced photosynthetic growth capabilities under dim-light conditions. The results of this study and additional studies indicate that regulated synthesis of the photosystem involves complex sets of overlapping regulatory circuits that differentially control photosystem gene expression in response to environmental stimuli such as oxygen tension and light intensity.     

A novel type of oxygenolytic ring cleavage: 2,4-oxygenation and decarbonylation of 1H-3-hydroxy-4-oxoquinaldine and 1H-3-hydroxy-4-oxoquinoline

Abstract The utilization of quinaldine (2-methylquinoline) by Arthrobacter sp. Rü61a proceeds via 1 H -4-oxoquinaldine, 1 H -3-hydroxy-4-oxoquinaldine, and N -acetyl-anthranilic acid. By analogy, 1 H -4-oxoquinoline is degraded by Pseudomonas putida 33/1 via 1 H -3-hydroxy-4-oxoquinoline and N -formylanthranilic acid. Using the purified enzymes from both organisms, the mode of N -heterocyclic ring cleavage was investigated. The conversions of 1 H -3-hydroxy-4-oxoquinaldine and 1 H -3-hydroxy-4-oxoquinoline to N -acetyl- and N -formylanthranilic acid, respectively, were both accompanied by the release of carbon monoxide. The enzyme-catalysed transformations were performed in an [¹⁸O]O₂ atmosphere and resulted in the incorporation of two oxygen atoms into the respective products, N -acetyl- and N -formylanthranilic acid, indicating an oxygenolytic attack at C-2 and C-4 of both 1 H -3-hydroxy-4-oxoquinaldine and 1 H -3-hydroxy-4-oxoquinolone.