AFLP marker polymorphism in cucumber (Cucumis sativus L.) near isogenic lines differing in sex expression

The AFLP technique was used to evaluate the level of polymorphism between two pairs of isogenic cucumber (Cucumis sativus L.) lines (NIL) differing in flower sex expression. The BSA techniques were also applied to find molecular markers linked to sex determination genes (dominant alleles) in those cucumber lines. Sex determination in cucumber is controlled by three main loci F, M and Gy. The interaction of these loci is responsible for the formation of the various phenotypes of flowers in respect to sex in the analyzed lines [corrected]. A female line 2gg with a ff/MM/gygy genotype, isogenic to a monoecious line B10 (genotype ff/MM/GyGy), and a female line Gy3 with a FF/MM/GyGy genotype, isogenic to a hermaphroditic line HGy3 (genotype FF/mm/GyGy). Using 56 combinations of AFLP primers, used for the analysis of lines 2gg and B10, gave 3794 bands, of which 155 (4.1%) were polymorphic. Ten bands distinguished gynoecious and monoecious bulks appearing at the same time in the appropriate parent; they are believed to be linked to the Gy locus. The isogenic lines Gy3 and HGy3 showed a higher level of polymorphism (14.2%). In this case, 55 combinations of primers gave 2996 reaction products, of which 430 showed variation. Twenty bands occurred in one bulk and in one parent, so they are probably associated with the M locus. Using the AFLP technique, the isogenicity of the lines was evaluated. The level of polymorphism (per pair of primer) between lines 2gg and B10 is 0.072% and is four times lower than that between the Gy3 and HGy3 lines (0.27%). The differences in the isogenicity of the lines can result from the degree of their relatedness, which may reflect the way they were derived.     

Increase in chloroplastic thiol groups by SO2 and its effect on light modulation of NADP-dependent glyceraldehyde 3-phosphate dehydrogenase

In broken spinach chloroplasts the total amount of thiol groups is about 3.7 mol mg^-1 chlorophyll. Two thirds are represented by the masked form (which is only titratable after unfolding of the protein). Of the free groups, those reacting with NBD·Cl (1.2–2.0 mol mg^-1 chlorophyll) seem to be undergoing oxidation more readily than those reacting with DTNB (1.0 mol mg^-1 chlorophyll). SO₂ application causes a maximal increase of 25% in free thiols, and doubles the amount of the masked thiols. The light triggered increase in SH, which starts at an elevated level, runs parallel to that of the controls. SO₂ application of 1.8 mg m^-3 (=28 nmol l^-1) for 1 h does not affect the dark level of NADP-GPD but enhances the light modulation by increasing the ratio of activation. This enhancement is explained by an increase in masked thiol groups during the preceding fumigation period.Abbreviations DTNB 5,5 dithiobis-2-nitrobenzene-2-oxa-1,3 diazole - NBD·Cl 7-chloro-4-nitrobenzene-2-oxa-1,3 diazole - PCMB p-chloromercuribenzoate - SDS sodium dodecylsulfate - NADP-GPD NADP-dependent glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.13) - HEPES N-2-Hydroxyethylpiperazine-N-2-ethanesulfonic acid - MES 2[N-Morpholino]ethanesulfonic acid - PGA 3-phosphoglyceric acid     

Conformations of the d-glucarolactones and d-glucaric acid in solution

The conformations of d-glucaric acid (1), d-glucaro-1,4-lactone (2), d-glucaro-6,3-lactone (3), and d-glucaro-1,4:6,3-dilactone (4) in solution were investigated by ¹H-n.m.r. and ¹³C-p.F.t., n.m.r. spectroscopy. The solvents used were deuterium oxide, methanol-d₄, and dimethyl sulfoxide-d₆, and praseodymium chloride was employed as a lanthanide shift-reagent. For 2, it was found that the conformational equilibrium ³E(d)

E₃(d) exists in solution, and that the OH-5 group tends to occupy the position over the lactone ring in the favored E₃(d),gg conformation. The n.m.r. data for 3 indicated that the conformational equilibrium is shifted in favor of the ⁴E(d)

E₄(d),gt conformation in solution. The dienvelope conformation ³E:E₄(d) was found to be the favored conformation of 4. For 1, a conformational equilibrium between one planar, zigzag form and two sickle forms was indicated by the n.m.r. data observed. ¹³C-N.m.r. spectroscopy proved to be a convenient method for monitoring the lactonization of 1, and the hydrolysis of its lactones. Lactones other than 2–4 were not found in solutions prepared from 1–4, either during their mutarotation or after equilibration at 30°.     

The Immunomodulatory Activity of Peptides Related to the DNA Contacting Loop of p53 Protein

Taking into account the sequence homology existing between thymopoietin II and the DNA-binding domain of p53 protein, a series of octapeptides was synthesized, related to the wild p53 type protein as well as to its mutated forms, appearing in some human tumours. The wild type octapeptide has immunostimulative activity with regard to the humoral immune response, but is inactive in the cellular immune response. The mutated peptides of p53 differ in their immunomodulatory activity from the wild type octapeptide. The Ser5 analogue of the wild type peptide is a strong stimulant of the humoral immune response and enhances TNF-α production, while at the same time suppressing the cellular immune response. The data suggest that the mutations of p53, which favour tumour development and growth, may also change the immune activity of respective p53 fragments.     

MNDO study of the mechanism of the inhibition of cysteine proteinases by diazomethyl ketones

Diazomethyl ketones are one of the most effective irreversible inhibitors of cysteine proteinases and are therefore very important in drug design. In the present study a mechanism of inactivation is proposed based on the results of model MNDO calculations of the possible pathways. It was found that the mercaptide nucleophile, on approaching the carbonyl carbon as in the catalytic reaction path, binds to the inner diazo nitrogen. The intermediate thus formed can rearrange giving a stable product, -thioketone, and molecular nitrogen, with a considerable energy gain. The energy barrier to this process is equal to 36.9 kcal/mol, and corresponds to a pyramidal transition state with the vertex at the methylene carbon and the base formed by the carbonyl, thiol, and diazo groups. The energy barrier can be lowered on deprotonation of the intermediate. Based on the results obtained it was concluded that good irreversible inhibitors of cysteine proteases must fulfil two structural requirements: i) the dimensions and charge distribution must be similar to those of the peptide bond and ii) a second electrophilic center must be present in the neighbourhood of the carbonyl carbon. These are requirements which are satisfied by other strong cysteine proteinase inhibitors: -chloroketones and -ketooxiranes.     

Differential translocation of protein kinase C isozymes by phorbol esters, EGF, and ANG II in rat liver WB cells

The protein kinaseC (PKC) family represents an important group of enzymes whoseactivation is associated with their translocation from the cytosol todifferent cellular membranes. In this study, the spatial distributionof PKC-, - and - in rat liver epithelial (WB) cells has beenexamined by Western blot analysis after subcellular fractionation.Cytosolic, membrane, nuclear, and cytoskeletal fractions were obtainedfrom cells stimulated with phorbol 12-myristate 13-acetate (PMA),angiotensin II (ANG II), or epidermal growth factor (EGF). PMA causedmost of the PKC-, - and - initially present in the cytosol tobe transported to the membrane and nuclear fractions. In contrast, bothANG II and EGF induced only a minor translocation of PKC- to themembrane fraction but caused a statistically significantmembrane-directed movement of PKC- and -. Translocation ofPKC- and - to the nucleus induced by ANG II and EGF was transient and quantitatively smaller than that induced by PMA. PKC- and -were present in the cytoskeleton of resting cells, but although PMA,ANG II, and EGF caused some changes in their content, these werevariable, suggesting that the cytoskeleton fraction was heterogeneous. PKC depletion inhibited ANG II-induced mitogenesis and the sustained activation of Raf-1 and extracellular regulated protein kinase (ERK).However, although PKC depletion inhibited EGF-induced mitogenesis, themaximum EGF-induced activation of the ERK pathway was only slightlyretarded. We hypothesize that PKC- and - are involved inmitogenesis via both ERK-dependent and ERK-independent mechanisms. These results support the notion that specific PKC isozymes exert spatially defined effects by virtue of their directed translocation todistinct intracellular sites.

     

Hydrogen Cycling by the Unicellular Marine Diazotroph Crocosphaera watsonii Strain WH8501

The hydrogen (H₂) cycle associated with the dinitrogen (N₂) fixation process was studied in laboratory cultures of the marine cyanobacterium Crocosphaera watsonii. The rates of H₂ production and acetylene (C₂H₂) reduction were continuously measured over the diel cycle with simultaneous measurements of fast repetition rate fluorometry and dissolved oxygen. The maximum rate of H₂ production was coincident with the maximum rates of C₂H₂ reduction. Theoretical stoichiometry for N₂ fixation predicts an equimolar ratio of H₂ produced to N₂ fixed. However, the maximum rate of net H₂ production observed was 0.09 nmol H₂ μg chlorophyll a (chl a)⁻¹ h⁻¹ compared to the N₂ fixation rate of 5.5 nmol N₂ μg chl a⁻¹ h⁻¹, with an H₂ production/N₂ fixation ratio of 0.02. The 50-fold discrepancy between expected and observed rates of H₂ production was hypothesized to be a result of H₂ reassimilation by uptake hydrogenase. This was confirmed by the addition of carbon monoxide (CO), a potent inhibitor of hydrogenase, which increased net H₂ production rates ∼40-fold to a maximum rate of 3.5 nmol H₂ μg chl a⁻¹ h⁻¹. We conclude that the reassimilation of H₂ by C. watsonii is highly efficient (>98%) and hypothesize that the tight coupling between H₂ production and consumption is a consequence of fixing N₂ at nighttime using a finite pool of respiratory carbon and electrons acquired from daytime solar energy capture. The H₂ cycle provides unique insight into N₂ fixation and associated metabolic processes in C. watsonii.The biological production of hydrogen (H₂) can occur as a by-product of photosynthesis, fermentation, and N₂ fixation (22). Of these three metabolic pathways, N₂ fixation remains a particularly enigmatic process, and to date there is no clear explanation for why H₂ evolves during the reduction of N₂ (11). The unfavorable energy cost of N₂ fixation can be mitigated by reassimilating the released H₂ via uptake hydrogenase enzyme activity (30). The coupled production and consumption of H₂ during cellular nitrogenase activity creates a H₂ cycle that can be hidden from measurements of ambient environmental H₂ concentrations and fluxes, depending upon the overall efficiency of H₂ assimilation (Fig. ​(Fig.11).Open in a separate windowFIG. 1.H₂ is formed during N₂ fixation by the binding of a N₂ molecule to the molybdenum-iron protein of the nitrogenase enzyme complex, prior to the reduction of N₂ to ammonia (11, 15). The most energetically favorable theoretical in vivo stoichiometry predicts that one mole of H₂ is produced for every mole of N₂ reduced: N₂ + 8H⁺ + 8e⁻ + 16ATP → 2NH₃ + H₂ + 16ADP + 16P_i. The production of H₂ consumes 25% of the electron flux through nitrogenase and diazotrophs mitigate this loss of potential energy by reassimilating the H₂ via uptake hydrogenase (21, 30). The electrons produced by uptake hydrogenase either generate reductant or ATP with simultaneous consumption of O₂ (3). (Adapted from reference 32a.)For most cultures of phototrophic marine diazotrophs grown under optimal conditions, complete reassimilation of H₂ is not achieved, and the excess H₂ is lost to the surrounding environment. This excess H₂ equates to the net production of H₂ and is expressed as the ratio of H₂ formed to N₂ fixed or the H₂/N₂ ratio. To date, H₂/N₂ ratios have mainly been measured on filamentous, colony-forming diazotrophs such as Anabaena spp. and Trichodesmium spp. with H₂ production rates of up to 20 nmol H₂ μg chlorophyll a (chl a)⁻¹ h⁻¹ and H₂/N₂ ratios ranging from 0.01 to 0.48 (3, 20, 24). H₂ production has also been quantified in unicellular diazotrophs (12, 16, 17, 32), although the H₂ measurements have rarely been performed in conjunction with rates of N₂ fixation. However, recent H₂ measurements of two N₂-fixing unicellular cyanobacteria species reached a maximum of 1.38 nmol H₂ μg chl a⁻¹ h⁻¹, with H₂/N₂ ratios ranging from 0.003 to 0.05, indicating an effective reassimilation of H₂ can occur under certain conditions (34).H₂ cycling in marine diazotrophs has important ecological implications both for the cell and for the marine H₂ cycle. Surface waters of low-latitude oceans are typically 200 to 300% supersaturated in dissolved H₂ with respect to atmospheric concentrations (25), implying a sustained localized production of H₂. The source of the dissolved H₂ is thought to be biological N₂ fixation (7); however, the relative contributions of diverse diazotrophic communities and in situ controls on H₂/N₂ ratios are not well constrained. N₂ fixation is performed by a suite of diazotrophs typically identified by their nitrogenase gene (nifH) sequences amplified directly from oceanic water samples (35). The importance of unicellular diazotrophs, including Crocosphaera spp., in marine N₂ fixation has recently become widely recognized (36). Size-fractionated rates of N₂ fixation indicate that in the oligotrophic ocean, <10-μm microorganisms, which include the unicellular cyanobacteria, make a substantial contribution to the daily N₂ fixation (9, 18). Correlating the species-specific production of H₂ with the activity and biomass of diazotrophs will help elucidate dissolved H₂ cycling in the upper ocean.We examined the cycling of H₂ in cultures of Crocosphaera watsonii strain WH8501, a marine unicellular diazotroph, and correlated it with other metabolic parameters, including N₂ fixation measured via acetylene (C₂H₂) reduction, O₂ production and consumption, and photosynthetic efficiency. Carbon monoxide (CO) was used as an inhibitor of intracellular H₂ reassimilation to reveal the H₂ cycling that can occur in conjunction with nitrogenase activity. H₂ reassimilation by C. watsonii was shown to be very efficient in our laboratory experiments, which is considered to be a consequence of the temporal separation between daytime photosynthetic activity and nighttime N₂ fixation. Therefore, the present study not only reveals the cell''s H₂ cycle but also provides insight into the metabolism of nitrogenase in C. watsonii.     

Near Scale-Free Dynamics in Neural Population Activity of Waking/Sleeping Rats Revealed by Multiscale Analysis

A neuron embedded in an intact brain, unlike an isolated neuron, participates in network activity at various spatial resolutions. Such multiple scale spatial dynamics is potentially reflected in multiple time scales of temporal dynamics. We identify such multiple dynamical time scales of the inter-spike interval (ISI) fluctuations of neurons of waking/sleeping rats by means of multiscale analysis. The time scale of large non-Gaussianity in the ISI fluctuations, measured with the Castaing method, ranges up to several minutes, markedly escaping the low-pass filtering characteristics of neurons. A comparison between neural activity during waking and sleeping reveals that non-Gaussianity is stronger during waking than sleeping throughout the entire range of scales observed. We find a remarkable property of near scale independence of the magnitude correlations as the primary cause of persistent non-Gaussianity. Such scale-invariance of correlations is characteristic of multiplicative cascade processes and raises the possibility of the existence of a scale independent memory preserving mechanism.     

The response of primary keratinocytes and fibroblasts from cancer patients to multiple low-dose irradiations

In our previous study, using the micronucleus (MN) assay, a hyper-radiosensitivity (HRS)-like phenomenon was observed after single low doses for fibroblasts from two and keratinocytes from four of the 40 patients studied. In this paper, we report the response of primary keratinocytes from 23 and fibroblasts from 21 of these cancer patients to multiple low-dose irradiations and answer the question regarding whether the patients with an HRS-like response after single low doses also demonstrate chromosomal hypersensitivity after multiple low doses. The cells were irradiated with three doses of 0.25 Gy separated by 4-h intervals, and MN induction was compared with that after the same total dose given as a single fraction of 0.75 Gy. Similarly, the effect of three doses of 0.5 Gy was compared with that of a single dose of 1.5 Gy. For fibroblasts from two and keratinocytes from four patients who demonstrated a single-dose HRS-like response, a significant inverse effect of fractionation (greater MN induction after three doses of 0.25 Gy than after a single dose of 0.75 Gy) was observed, which suggests a repeated hypersensitive response after each dose of 0.25 Gy. Such an effect was not seen for the cells from 19 patients who were single-dose HRS-like negative. In conclusion, an inverse fractionation effect for MN induction that was observed in fibroblasts from two and keratinocytes from four patients after three doses of 0.25 Gy (but not 3 x 0.5 Gy) reflects the chromosomal hyper-radiosensitivity seen in the same patients in response to single low doses.