Ethylene synthesis and auxin augmentation in pistil tissues are important for egg cell differentiation after pollination in maize

The role of ethylene and auxin in stigma-to-ovule signalling was investigated in maize (Zea mays L.). Maturation of the egg cells in an ear was stimulated before actual fertilization by the application of fresh pollen grains or quartz sand to fully receptive stigmas. Ethylene emission by maize ears increased in response to those treatments. Silks and ovaries were involved in ethylene synthesis after pollen or sand was shed over the silks. The content of ethylene precursor [1-aminocyclopropane-1-carboxylic acid (ACC)] increased in both pistil parts soon after pollination. ACC rise was delayed by 4 h in the ovaries, and by 8 h in the silks after mock-pollination with sand. The auxin level increased rapidly in the silks and ovaries after pollination, and it was very high in the pollinated silks due to the high indole-3-acetic acid (IAA) content of pollen grains. IAA rise also appeared in the silks and ovaries after treatment with sand but it was delayed by 8 h. Application of ACC (10 microM) or IAA (6 microM) solutions to non-pollinated silks stimulated maturation of the egg cells. Moreover, the response of the egg cells to pollination was cancelled by l-alpha-(2-aminoethoxyvinyl)-glycine, alpha-aminoisobutyric acid or 2,3,5-triiodobenzoic acid applied to the silks before pollination. Thus ethylene synthesis and polar auxin transport in the silks pollinated with fresh pollen were necessary to evoke accelerated differentiation of the egg cells in maize ovules. Differences in pistil responses found between true- and mock-pollination suggest that signalling pathways are at least partially different for the reception of pollen grains and sand crystals on maize stigma.     

Homogeneity and distinctiveness of Polish paternal lineages revealed by Y chromosome microsatellite haplotype analysis

Different regional populations from Poland were studied in order to assess the genetic heterogeneity within Poland, investigate the genetic relationships with other European populations and provide a population-specific reference database for anthropological and forensic studies. Nine Y-chromosomal microsatellites were analysed in a total of 919 unrelated males from six regions of Poland and in 1,273 male individuals from nine other European populations. AMOVA revealed that all of the molecular variation in the Polish dataset is due to variation within populations, and no variation was detected among populations of different regions of Poland. However, in the non-Polish European dataset 9.3% ( P<0.0001) of the total variation was due to differences among populations. Consequently, differences in R(ST)-values between all possible pairs of Polish populations were not statistically significant, whereas significant differences were observed in nearly all comparisons of Polish and non-Polish European populations. Phylogenetic analyses demonstrated tight clustering of Polish populations separated from non-Polish groups. Population clustering based on Y-STR haplotypes generally correlates well with the geography and history of the region. Thus, our data are consistent with the assumption of homogeneity of present-day paternal lineages within Poland and their distinctiveness from other parts of Europe, at least in respect to their Y-STR haplotypes. Electronic supplementary material to this paper can be obtained by using the Springer LINK server located at http://dx.doi.org/10.1007/s00439-002-0728-0.     

Third calcium-modulated rod outer segment membrane guanylate cyclase transduction mechanism

Ca²⁺-modulated rod outer segment membrane guanylate cyclase (ROS-GC1) has been cloned and reconstituted to show that it is regulated by two processes: one inhibitory, the other stimulatory. The inhibitory process is consistent with its linkage to phototransduction; the physiology of the stimulatory process is probably linked to neuronal transmission. In both regulatory processes, calcium modulation of the cyclase takes place through the calcium binding proteins; guanylate cyclase activating proteins (GCAP1 and GCAP2) in the case of the phototransduction process and calcium-dependent GCAP (CD-GCAP) in the case of the stimulatory process. The cyclase domains involved in the two processes are located at two different sites on the ROS-GC1 intracellular region. The GCAP1-modulated domain resides within the aa 447-730 segment of ROS-GC1 and the CD-GCAP-modulated domain resides within the aa 731-1054 segment. In the present study the GCAP2-dependent Ca²⁺ modulation of the cyclase activity has been reconstituted using recombinant forms of GCAP2 and ROS-GC1, and its mutants. The results indicate that consistent to phototransduction, GCAP2 at low Ca²⁺ concentration (10 nM) maximally stimulates the cyclase activity of the wild-type and its mutants: ext- (deleted aa 8-408); kin- (deleted aa 447-730) and hybrid consisting of the ext, transmembrane and kin domains of ANF-RGC and the C-terminal domain, aa 731-1054, of ROS-GC1. In all cases, it inhibits the cyclase activity with an IC₅₀ of about 140 nM. A previous study has shown that under identical conditions the kin- and the hybrid mutant are at best only minimally stimulated. Thus, the GCAP1 and GCAP2 signal transduction mechanisms are different, occurring through different modules of ROS-GC1. These findings also demonstrate that the intracellular region of ROS-GC1 is composed of multiple modules, each designed to mediate a particular calcium-specific signalling pathway.     

The kinetics of cytological events during double fertilization in Zea mays L.

By using a clearing method, the process of double fertilization in Zea mays L. (line A 188) was analysed and the precise sequence of events was determined. The period from pollen tube arrival to gamete fusion was relatively short, possibly less than 1 h. The karyogamy was of premitotic type, and the time from the contact of male and female nuclei to the fusion of male and female nucleoli was about 5 h in the egg cell and 3 h in the central cell. In the central cell, the sperm nucleus fused with either one of the polar nuclei or the secondary nucleus, the latter being observed for the first time in maize. The zygote was in the resting period for 13–16 h before division commenced, changing the cell polarity during karyogamy and the resting period. The primary endosperm nucleus divided immediately after karyogamy was completed in the central cell. The embryo sacs with two-celled proembryos contained four to eight endosperm nuclei. The timetable of fertilization events could be a standard for further studies on in vitro fertilization at the cytological and molecular levels.     

Synthesis and biological activity of 22-iodo- and (E)-20(22)-dehydro analogues of 1α,25-dihydroxyvitamin D3

Construction of 25-hydroxy-steroidal side chain substituted with iodine at C-22 was elaborated on a model PTAD-protected steroidal 5,7-diene and applied to a synthesis of (22R)- and (22S)-22-iodo-1α,25-dihydroxyvitamin D₃. Configuration at C-22 in the iodinated vitamins, obtained by nucleophilic substitution of the corresponding 22S-tosylates with sodium iodide, was determined by comparison of their iodine-displacement processes and cyclizations leading to isomeric five-membered (22,25)-epoxy-1α-hydroxyvitamin D₃ compounds. Also, 20(22)-dehydrosteroids have been obtained and their structures established by ¹H NMR spectroscopy. When compared to the natural hormone, (E)-20(22)-dehydro-1α,25-dihydroxyvitamin D₃ was found 4 times less potent in binding to the porcine intestinal vitamin D receptor (VDR) and 2 times less effective in differentiation of HL-60 cells. 22-Iodinated vitamin D analogues showed somewhat lower in vitro activity, whereas (22,25)-epoxy analogues were inactive. Interestingly, it was established that (22S)-22-iodo-1α,25-dihydroxyvitamin D₃ was 3 times more potent than its (22R)-isomer in binding to VDR and four times more effective in HL-60 cell differentiation assay. The restricted mobility of the side chain of both 22-iodinated vitamin D compounds was analyzed by a systematic conformational search indicating different spatial regions occupied by their 25-oxygen atoms. Preliminary data on the in vivo calcemic activity of the synthesized vitamin D analogues indicate that (E)-20(22)-dehydro-1α,25-dihydroxyvitamin D₃ and 22-iodo-1α,25-dihydroxyvitamin D₃ isomers were ca. ten times less potent than the natural hormone 1α,25-(OH)₂D₃ both in intestinal calcium transport and bone calcium mobilization.     

The Psm locus controls paternal sorting of the cucumber mitochondrial genome

The mitochondrial genome of cucumber shows paternal transmission and there are no reports of variation for mitochondrial transmission in cucumber. We used a mitochondrially encoded mosaic (MSC) phenotype to reveal phenotypic variation for mitochondrial-genome transmission in cucumber. At least 10 random plants from each of 71 cucumber plant introductions (PIs) were crossed as the female with an inbred line (MSC16) possessing the MSC phenotype. Nonmosaic F1 progenies were observed at high frequencies (greater than 50%) in F1 families from 10 PIs, with the greatest proportions being from PI 401734. Polymorphisms near the mitochondrial cox1 gene and JLV5 region revealed that nonmosaic hybrid progenies from crosses of PI 401734 with MSC16 as the male possessed the nonmosaic-inducing mitochondrial DNA (mtDNA) from the paternal parent. F2) F3, and backcross progenies from nonmosaic F1 plants from PI 401734 x MSC16 were testcrossed with MSC16 as the male parent to reveal segregation of a nuclear locus (Psm for Paternal sorting of mitochondria) controlling sorting of mtDNA from the paternal parent. Psm is a unique locus at which the maternal genotype affects sorting of paternally transmitted mtDNA.     

Phospholipase D elevates the level of MDM2 and suppresses DNA damage-induced increases in p53

Phospholipase D (PLD) has been reported to generate survival signals that prevent apoptosis induced by serum withdrawal. We have now found that elevated expression of PLD also suppresses DNA damage-induced apoptosis. Since DNA damage-induced apoptosis is often mediated by p53, we examined the effect of elevated PLD expression on the regulation of p53 stabilization. We report here that PLD suppresses DNA damage-induced increases in p53 stabilization in cells where PLD has been shown to provide a survival signal. Elevated expression of PLD also led to increased expression of the p53 E3 ubiquitin ligase MDM2 and increased turnover of p53. PLD1-stimulated increases in MDM2 expression and suppression of p53 activation were blocked by inhibition of mTOR and the mitogen-activated protein kinase pathway. Although PLD did not activate the phosphatidylinositol 3-kinase (PI3K)/Akt survival pathway activate the basal levels of PI3K activity were partially required for PLD1-induced increases in MDM2. These data provide evidence that survival signals generated by PLD involve suppression of the p53 response pathway.     

ATX-2, the C. elegans ortholog of ataxin 2, functions in translational regulation in the germline

Human ataxin 2 is a protein of unknown function that is implicated in the neurodegenerative disorder spinocerebellar ataxia type 2. We found that the C. elegans ortholog of ataxin 2, ATX-2, forms a complex with PAB-1, a cytoplasmic polyA-binding protein, and that ATX-2 is required for development of the germline. In the absence of ATX-2, proliferation of stem cells is reduced, and the germline is abnormally masculinized. These defects appear to result from inappropriate translational regulation that normally is mediated by the conserved KH-domain protein GLD-1. We find that MEX-3, a second KH-domain protein, exhibits a novel, ATX-2-dependent role in preventing inappropriate translation in the germline stem cells. Together, our results suggest that ATX-2 functions in translational regulation that is mediated by GLD-1 and MEX-3 proteins.     

New derivative of 1alpha,25-dihydroxy-19-norvitamin D3 with 3'-alkoxypropylidene moiety at C-2: synthesis, biological activity and conformational analysis

In pursuit of novel biologically active Vitamin D compounds of potential therapeutic value, 1alpha,25-dihydroxy-2-[3'-(methoxymethoxy)propylidene]-19-norvitamin D(3) (7) was efficiently prepared in a convergent synthesis, starting with (-)-quinic acid and the protected 25-hydroxy Grundmann ketone 16. The key synthetic step involved Lythgoe type Wittig-Horner coupling of 16, with the phosphine oxide 15. Molecular modeling was employed to establish the A-ring conformation of the synthesized Vitamin 7. Also, preliminary modeling of its complex with the rVDR was performed and interactions between ligand and the binding domain analyzed. Analog 7 was found to be only six times less potent than 1alpha,25-(OH)(2)D(3) (1) in binding to the rat recombinant Vitamin D receptor (VDR). In comparison with hormone 1, it also showed slightly lower cellular HL-60-differentiation activity. Preliminary in vivo tests indicated unusually high calcemic activity of 7.