Genotype, ploidy and physiological state in relation to isoperoxidases in Nicotiana

Peroxidase and isoperoxidase patterns of tobacco have been comparatively analyzed in relation to parental origin and ploidy level (including hypohaploidy status) at different ages and physiological states. The interspecific hybrid N. sylvestris × N. tomentosiformis shows an additive pattern of parental peroxidases, which also resembles that of N. tabacum cv. Wisconsin and cv. Samsun. Enzyme activity (on dry weight and protein bases, decreases from the diploid to the hypohaploid forms of Wisconsin and Samsun with respective quantitative differences in the zymograms, when the plants are compared at the same chronological and vegetative stages. Abnormally vegetatively ageing hypohaploids recover all peroxidasic isoenzymes and reach an enzyme activity as high as those of haploids and diploids. Flowering hypohaploids, however, generally behave like haploids and diploids in changing their balance between acidic and basic peroxidases.     

The matrix metalloproteinase pump-1 catalyzes formation of low molecular weight (pro)urokinase in cultures of normal human kidney cells.

The enzyme responsible for the metalloproteinase activity which cleaves the Glu143-Leu144 bond of (pro)urokinase has been isolated from the conditioned medium of cultured normal human kidney cells. Using S-Sepharose and Cibacron Blue-agarose chromatography, then C-4 reversed phase high pressure liquid chromatography, a protein of about 20,000 Da was isolated. Through an identical amino-terminal sequence, the protein was shown to be the matrix metalloproteinase previously referred to in the literature as "pump-1" (putative metalloproteinase). When aprotinin was added during the course of the purification, the major species isolated was the zymogen form (28,000 Da) of pump-1. Pump-1 has been shown to efficiently cleave the susceptible bond of both pro-urokinase (single-chain) and active (two-chain) urokinase and thereby produce the corresponding low molecular weight forms. The amino-terminal sequences of the A and B chains of low molecular weight urokinase prepared by action of pump-1 on recombinant high molecular weight urokinase are identical to those of the low molecular weight urokinase isolated from human kidney cell culture. Since the reaction of urokinase with this metalloproteinase results in separation of its serine proteinase region from the domain which mediates binding to the urokinase receptor, it may be of importance in the regulation of the functional activity of the plasminogen activator in cellular processes.     

Studies with mechanism-based inactivators of lysine epsilon-transaminase from Achromobacter liquidum.

Analogues of lysine containing a 4,5-acetylenic linkage (lysyne) or a cis- or trans-4,5-olefinic linkage (lysenes) function as substrates for a homogeneous L-lysine epsilon-transaminase from Achromobacter liquidum but partition between transamination and time-dependent inactivation. The partition ratio is lowest for lysyne (40 per inactivation event) and higher for trans-lysene (160 per inactivation event), and the cis-lysene transaminates 1600 times per inactivation event. cis-Lysene yields alpha-picolinate as a detectable accumulating product, presumably from cyclization of initial 6-aldehyde to dihydropicolinate and spontaneous autoxidation. The trans isomer also yields some picolinate as an identifiable product. The product from the few lysyne turnovers is as yet unknown but has strong absorbance at 318 nm. The inactive enzyme species from all three lysine analogues slowly (overnight) regain full activity after gel filtration chromatography and dialysis, suggesting reversal of the initial adduct-forming reaction. Initial studies with partially purified pseudomonad lysine alpha-racemase show alpha-3H incorporation from 3H2O but no inactivation consistent with the expectation that these lysine analogues could act readily as mechanism-based inactivators for pyridoxal P enzymes which act at the epsilon- but not the alpha-carbon of lysine.     

A multidimensional 1H NMR investigation of the conformation of methionine-enkephalin in fast-tumbling bicelles

Enkephalins are pentapeptides found in the central nervous system. It is believed that these neuropeptides interact with the nerve cell membrane to adopt a conformation suitable for their binding to an opiate receptor. In this work, we have determined the three-dimensional structure of methionine-enkephalin (Menk) in fast-tumbling bicelles using multidimensional (1)H NMR. Bicelles were selected as model membranes because both their bilayer organization and composition resemble those of natural biomembranes. The effect of the membrane composition on the peptide conformation was explored using both zwitterionic (PC bicelles) and negatively charged bicelles (Bic/PG). Pulsed field gradient experiments allowed the determination of the proportion of Menk bound to the model membranes. Approximately 60% of the water-soluble enkephalin was found to associate to the bicellar systems. Structure calculations from torsion angle and NOE-based distance constraints suggest the presence of both micro - and delta-selective conformers of Menk in each system and slightly different conformers in PC bicelles and Bic/PG. As opposed to previous studies of enkephalins in membrane mimetic systems, our results show that these opiate peptides could adopt several conformations in a membrane environment, which is consistent with the flexibility and poor selectivity of enkephalins.     

Differential protein acetylation induced by novel histone deacetylase inhibitors

Histone deacetylase (HDAC) inhibitors induce the hyperacetylation of nucleosomal histones in carcinoma cells resulting in the expression of repressed genes that cause growth arrest, terminal differentiation, and/or apoptosis. In vitro selectivity of several novel hydroxamate HDAC inhibitors including succinimide macrocyclic hydroxamates and the non-hydroxamate alpha-ketoamide inhibitors was investigated using isolated enzyme preparations and cellular assays. In vitro selectivity for the HDAC isozymes (HDAC1/2, 3, 4/3, and 6) was not observed for these HDAC inhibitors or the reference HDAC inhibitors, MS-275 and SAHA. In T24 and HCT116 cells these compounds caused the accumulation of acetylated histones H3 and H4; however, the succinimide macrocyclic hydroxamates and the alpha-ketoamides did not cause the accumulation of acetylated alpha-tubulin. These data suggest "selectivity" can be observed at the cellular level with HDAC inhibitors and that the nature of the zinc-chelating moiety is an important determinant of activity against tubulin deacetylase.     

Insights on the interaction of met-enkephalin with negatively charged membranes--an infrared and solid-state NMR spectroscopic study

Enkephalins are pentapeptides found in the human nervous system, where they are involved in the relief of pain. The interaction of these neuropeptides with the nerve cell membranes would be a key-step in the receptor binding. We have used both Fourier-transform infrared and solid-state NMR spectroscopies to shed light on the interactions responsible for the association of enkephalins with negatively charged membranes. More specifically, we have investigated the interaction of methionine-enkephalin (Menk) with DMPG and DMPS vesicles. Our results suggest that Menk interacts electrostatically with both model membranes via its terminal NH₃⁺ group. However, the peptide induced the formation of elongated DMPG vesicles in the magnetic field. On the other hand, the association of Menk with DMPS bilayers was concentration-dependent and disrupted the membrane at high peptide concentrations. The different effect of methionine-enkephalin with the two types of anionic membranes is most likely related to the different fluidity of these systems.     

Impact of male mating history on the temporal sperm dynamics of Choristoneura rosaceana and C. fumiferana females

In the oblique-banded leafroller, Choristoneura rosaceana, and the spruce budworm, C. fumiferana, male reproductive performance decreases with consecutive matings. While the onset time of mating did not vary, the time spent mating was longer in mated than in virgin males. Furthermore, a decline observed in the spermatophore mass with successive matings was associated with a concomitant decline in its apyrene and eupyrene spermatozoa content. In the hours following mating, spermatozoa migrate from the spermatophore, located in the bursa copulatrix, to the spermatheca. Regardless of the male's previous mating history, the number of apyrene sperm dropped rapidly in the days following mating whereas the number of eupyrene spermatozoa declined gradually. As the temporal pattern of sperm movement was similar in all treatments, females mated with previously-mated males would suffer from sperm shortage sooner than those mated with virgins. Large C. rosaceana females stored more apyrene spermatozoa in their spermatheca than small ones, irrespective of the time after mating or male mating history, while only large females mated with once-mated males received more apyrene sperm and accessory gland secretions than small ones mated with virgin or twice-mated males. The results obtained in this study are discussed in relation with their potential impact on the reproductive success of both sexes.     

A highly parallel strategy for storage of digital information in living cells

Background

Encoding arbitrary digital information in DNA has attracted attention as a potential avenue for large scale and long term data storage. However, in order to enable DNA data storage technologies there needs to be improvements in data storage fidelity (tolerance to mutation), the facility of writing and reading the data (biases and systematic error arising from synthesis and sequencing), and overall scalability.

Results

To this end, we have developed and implemented an encoding scheme that is suitable for detecting and correcting errors that may arise during storage, writing, and reading, such as those arising from nucleotide substitutions, insertions, and deletions. We propose a scheme for parallelized long term storage of encoded sequences that relies on overlaps rather than the address blocks found in previously published work. Using computer simulations, we illustrate the encoding, sequencing, decoding, and recovery of encoded information, ultimately demonstrating the possibility of a successful round-trip read/write. These demonstrations show that in theory a precise control over error tolerance is possible. Even after simulated degradation of DNA, recovery of original data is possible owing to the error correction capabilities built into the encoding strategy. A secondary advantage of our method is that the statistical characteristics (such as repetitiveness and GC-composition) of encoded sequences can also be tailored without sacrificing the overall ability to store large amounts of data. Finally, the combination of the overlap-based partitioning of data with the LZMA compression that is integral to encoding means that the entire sequence must be present for successful decoding. This feature enables inordinately strong encryptions. As a potential application, an encrypted pathogen genome could be distributed and carried by cells without danger of being expressed, and could not even be read out in the absence of the entire DNA consortium.

Conclusions

We have developed a method for DNA encoding, using a significantly different fundamental approach from existing work, which often performs better than alternatives and allows for a great deal of freedom and flexibility of application.

     

Drought Rhizogenesis in Arabidopsis thaliana (Differential Responses of Hormonal Mutants)

Drought rhizogenesis is an adaptive strategy that occurs during progressive drought stress and is characterized in the Brassicaceae and related families by the formation of short, tuberized, hairless roots. These roots are capable of withstanding a prolonged drought period and give rise to a new functional root system upon rehydration. The kinetics of drought rhizogenesis during progressive water shortage was analyzed in the Arabidopsis thaliana wild-type ecotypes Landsberg erecta and Columbia. In both genotypes, this response started from a similar threshold of soil humidity (about 2%). The intensity of drought rhizogenesis was compared in various A. thaliana hormonal mutants. The wild-type lines and most of the mutants achieved a similiar drought rhizogenetic index (DRI), defined as the maximum number of short roots produced per mg of root biomass, after progressive drought stress. However, this DRI was dramatically reduced in the abscisic acid (ABA)-deficient aba, ABA-insensitive abi1-1, and auxin-resistant axr1-3 mutants. These data indicate that endogenous ABA and auxin play a promotive role in drought rhizogenesis. The DRI was highly increased in the gibberellin (GA) biosynthetic mutant ga5, suggesting that some GAs might also participate in this process. The possible role and identity of the GA species involved is discussed in view of the unaltered DRI values of the ga2, ga3, and ga4 mutants. The present analysis also allowed further discrimination among the various ABA-insensitive (abi1 versus abi2 and abi3) and auxin-resistant (axr1 versus aux1) mutants tested. In particular, drought rhizogenesis is the first physiological response shown to be differentially affected by the abi1-1 and abi2-1 mutations.     

Chromosomal location of genes controlling short-term and long-term somatic embryogenesis in wheat revealed by immature embryo culture of aneuploid lines

The expression of essential genes during somatic embryogenesis can be analysed by inducing aneuploid cells to undergo embryogenesis during immature embryo culture and then determining whether defects occur. Triticum aestivum disomic and aneuploid stocks, including 36 ditelosomics and 7 nullitetrasomic Chinese Spring wheats, were compared for their ability to undergo somatic embryogenesis after 2 months of in vitro immature embryo culture. Their regeneration capacity was observed after 4 and 14 months of in vitro culture to determine which chromosome arms influence the process. The large range of variation found among the tested aneuploids suggested that genetic control of the somatic tissue culture ability is polygenic. Our results indicate that genes affecting somatic embryo-genesis and regeneration are located in all of the homoeologous chromosome groups. The lack of chromosome arms 1AL (DT 1AS) and 3DL (DT 3DS) practically suppresses somatic embryogenesis, demonstrating that major genes on wheat chromosome arms 1AL and 3DL control regeneration capacity. Results suggest that plants were mainly produced from somatic embryo development. Although the control of somatic embryogenesis and regeneration is polygenic, the genes located on the long arms of homoeologous group 3 chromosomes have a major effect. We also have evidence of chromosome arms that determine the time required for regeneration.