ASY1 coordinates early events in the plant meiotic recombination pathway.

Meiosis is a fundamental and evolutionarily conserved process that is central to the life cycles of all sexually reproducing eukaryotes. An understanding of this process is critical to furthering research on reproduction, fertility, genetics and breeding. Plants have been used extensively in cytogenetic studies of meiosis during the last century. Until recently, our knowledge of the molecular and functional aspects of meiosis has emerged from the study of non-plant model organisms, especially budding yeast. However, the emergence of Arabidopsis thaliana as the model organism for plant molecular biology and genetics has enabled significant progress in the characterisation of key genes and proteins controlling plant meiosis. The development of molecular and cytological techniques in Arabidopsis, besides allowing investigation of the more conserved aspects of meiosis, are also providing insights into features of this complex process which may vary between organisms. This review highlights an example of this recent progress by focussing on ASY1, a meiosis-specific Arabidopsis protein which shares some similarity with the N-terminus region of the yeast axial core-associated protein, HOP1, a component of a multiprotein complex which acts as a meiosis-specific barrier to sister-chromatid repair in budding yeast. In the absence of ASY1, synapsis is interrupted and chiasma formation is dramatically reduced. ASY1 protein is initially detected during early meiotic G2 as numerous foci distributed over the chromatin. As G2 progresses the signal appears to be increasingly continuous and is closely associated with the axial elements. State-of-the-art cytogenetic techniques have revealed that initiation of recombination is synchronised with the formation of the chromosome axis. Furthermore, in the context of the developing chromosome axes, ASY1 plays a crucial role in co-ordinating the activity of a key member of the homologous recombination machinery, AtDMC1.

Control of meiotic recombination in Arabidopsis: role of the MutL and MutS homologues.

Immunocytochemistry reveals that the Arabidopsis mismatch repair proteins AtMSH4, AtMLH3 and AtMLH1 are expressed during prophase I of meiosis. Expression of AtMSH4 precedes AtMLH3 and AtMLH1 which co-localize as foci during pachytene. Co-localization between AtMSH4 and AtMLH3 occurs, but appears transient. AtMLH3 foci are not detected in an Atmsh4 mutant. However, localization of AtMSH4 is unaffected in Atmlh3, suggesting that recombination may proceed to dHj (double Holliday junction) formation. Mean chiasma frequency in Atmsh4 is reduced to 1.55 compared with 9.86 in wild-type. In contrast with wild-type, the distribution of residual crossovers in Atmsh4 closely fits a Poisson distribution. This is consistent with a two-pathway model for meiotic crossing-over whereby most crossovers occur via an AtMSH4-dependent pathway that is subject to interference, with the remaining crossovers arising via an interference-independent pathway. Loss of AtMLH3 results in an approx. 60% reduction in crossovers. Results suggest that dHj resolution can occur, but in contrast with wild-type where most or all dHjs are directed to form crossovers, the outcome is biased in favour of a non-crossover outcome. The results are compatible with a model whereby the MutL complex maintains or imposes a dHj conformation that ensures crossover formation.

A strategy to investigate the plant meiotic proteome.

The analysis of meiosis in higher plants has benefited considerably in recent years from the completion of the genome sequence of the model plant Arabidopsis thaliana and the development of cytological techniques for this species. A combination of forward and reverse genetics has provided important routes toward the identification of meiotic genes in Arabidopsis. Nevertheless identification of certain meiotic genes remains a challenge due to problems such as limited sequence conservation between species, existence of closely related gene families and in some cases functional redundancy between gene family members. Hence there is a requirement to develop new experimental approaches that can be used in conjunction with existing methods to enable a greater range of plant meiotic genes to be identified. As one potential route towards this goal we have initiated a proteomics-based approach. Unfortunately, the small size of Arabidopsis anthers makes an analysis in this species technically very difficult. Therefore we have initially focussed on Brassica oleracea which is closely related to Arabidopsis, but has the advantage of possessing significantly larger anthers. The basic strategy has been to use peptide mass-finger printing and matrix-assisted laser desorption ionization time of flight mass spectrometry to analyse proteins expressed in meiocytes during prophase I of meiosis. Initial experiments based on the analysis of proteins from staged anther tissue proved disappointing due to the low level of detection of proteins associated with meiosis. However, by extruding meiocytes in early prophase I from individual anthers prior to analysis a significant enrichment of meiotic proteins has been achieved. Analysis suggests that at least 18% of the proteins identified by this route have a putative meiotic function and that this figure could be as high as one-third of the total. Approaches to increase the enrichment of proteins involved in meiotic recombination and chromosome synapsis are also described.

RP463: a stabilized technetium-99m complex of a hydrazino nicotinamide derivatized chemotactic peptide for infection imaging.

A HYNIC-conjugated chemotactic peptide (fMLFK-HYNIC) was labeled with (99m)Tc using tricine and TPPTS as coligands. The combination of fMLFK-HYNIC, tricine, and TPPTS with (99m)Tc produced a ternary ligand complex [(99m)Tc(fMLFK-HYNIC)(tricine)(TPPTS)] (RP463). RP463 was synthesized either in two steps, in which the binary ligand complex [(99m)Tc(fMLFK-HYNIC)(tricine)(2)] (RP469) was formed first and then reacted with TPPTS, or in one step by direct reduction of [(99m)Tc]pertechnetate with stannous chloride in the presence of fMLFK-HYNIC, tricine, and TPPTS. The radiolabeling yield for RP463 was usually >/=90% using 10 microg of fMLFK-HYNIC and 100 mCi of [(99m)Tc]pertechnetate. Unlike RP469, which decomposed rapidly in the absence of excess tricine coligand, RP463 was stable in solution for at least 6 h. [(99)Tc]RP463 was prepared and characterized by HPLC and electrospray mass spectrometry. In an in vitro assay, [(99)Tc]RP463 showed an IC(50) of 2 nM against binding of [(3)H]fMLF to receptors on PMNs. [(99)Tc]RP463 also induces effectively the superoxide release of polymorphonuclear leukocytes (PMNs) with an EC(50) value of 0.2 +/- 0.2 nM. The localization of RP463 in the infection foci was assessed in a rabbit infection model. RP463 was cleared from the blood faster than RP469 and was excreted mainly through the renal system. As a result of rapid blood clearance and increased uptake, the target-to-background ratios continuously increased from 1.5 +/- 0.2 at 15 min postinjection to 7.5 +/- 0.4 at 4 h postinjection. Visualization of the infected area could be as early as 2 h. A transient decrease in white blood cell count of 35% was observed during the first 30 min after injection of the HPLC-purified RP463 in the infected rabbit. This suggests that future research in this area should focus on developing highly potent antagonists for chemotactic peptide receptor or other receptors on PMNs and monocytes.

Selective inhibition of N-formylpeptide-induced neutrophil activation by carbamate-modified peptide analogues.

Stimulation of the leukocyte N-formylpeptide receptor (FPR) induces chemotaxis, cell adhesion, free radical release, and degranulation, responses associated with infection and inflammation. Under conditions where continuous activation of the receptor prevails, neutrophil-dependent tissue damage ensues. Antagonists of the FPR have potential for use as diagnostic and therapeutic agents. Hence, we have synthesized and evaluated a series of amino-terminal carbamate analogues of the peptide Met-Leu-Phe (MLF) in order to determine the structural requirements for imparting agonist or antagonist activity at the human neutrophil FPR. Peptides were evaluated in three in vitro assays: receptor binding, superoxide anion release, and cell adhesion. Unbranched carbamates (methoxycarbonyl, ethoxycarbonyl, and n-butyloxycarbonyl) resulted in agonist activity, whereas branched carbamates (iso-butyloxycarbonyl, tert-butyloxycarbonyl, and benzyloxycarbonyl) were antagonists. The peptide antagonists were more potent inhibitors of superoxide anion release than cell adhesion by 4-7-fold. When iso-butyloxycarbonyl-MLF (i-Boc-MLF) was further modified at the carboxy terminus with Lys, antagonist potency was retained but without functional selectivity. Further C-terminal modification with the radionuclide linker diethylenetriaminepentaacetic acid did not alter the potency of i-Boc-MLFK. These results indicate that the switch from agonist to antagonist activity can be achieved by modifying the overall size and shape of the amino-terminal group; that modifications at both the amino and carboxy termini can alter the functional selectivity of the peptide; and that modifications can be tolerated at the carboxy terminus to allow for development of an antagonist for diagnostic applications.

Synthesis and cytotoxicity of some cyclometallated palladium complexes.

Several monomeric cyclometallated palladium complexes have been prepared by bridge opening reactions of their corresponding dimeric precursors with various amines. The complexes were characterized by elemental analyses and proton NMR spectroscopy. The general formula for the complexes can be given as Pd(N-C)LX where (N-C) is an aromatic or aliphatic amine coordinated as a chelating ligand through the amine and a formal cyclometallated Pd-C bond; L = amine, X = chloride or acetate. A unique complex based on the cyclopalladation of 2-phenyl phenanthroline was also prepared directly. The series of complexes was screened for cytotoxicity against a panel of seven human tumor cell lines. All complexes were found to be cytotoxic (IC50) at micrograms/ml concentrations, while two complexes also displayed some differential cytotoxicity.

Platinum and other metal coordination compounds in cancer chemotherapy. A commentary on the sixth international symposium: San Diego, California, 23-26th January 1991.

The use of molecular biological methodologies has provided a greater understanding of the cytotoxic effects of cisplatin and the underlying mechanisms of tumour cell resistance. Resistance to cisplatin is often multifocal with plasma membrane, cytosolic and nuclear components. Cisplatin-DNA adducts appear to be recognised by specific damage recognition proteins. Proteins associated with the transport of platinum through plasma membranes and genes associated with cisplatin resistance appear to be close to being elucidated. Current Phase I and Phase II clinical trials with platinum-containing complexes largely focus on the 1,2 diaminocyclohexane (DACH) carrier ligand, the dicarboxylatocyclobutane leaving group and complexes which circumvent cisplatin resistance in murine leukaemia models. At present, the trials are at too early a stage to allow comment on their clinical utility and, consequently, the relevance of the murine leukaemia-based preclinical observations. On the horizon, orally active platinum (IV) ammine/amine dicarboxylate dichloride coordination complexes with preclinical toxicological profiles similar to carboplatin should enter clinical trial in the next year.

Cardiac activity preparatory to overt and covert behavior.

The heart rates of 17 women subjects were recorded as they prepared to make both overt (key press) and covert (silently thinking the word "stop") responses. A very reliable preparatory cardiac response was obtained regardless of whether the overt or covert response mode was employed. The temporal development of this cardiac response faithfully reflected the speed with which the subjects were asked to respond, suggesting that in the covert condition heart rate could be used to detect the time at which a mental event was being generated.