Identification of Stylar RNases Associated with Gametophytic Self-Incompatibility in Almond (Prunus dulcis)

Stylar proteins of 13 almond (Prunus dulcis) cultivars withknown S-genotypes were surveyed by IEF and 2D-PAGE combinedwith immunoblot and N-terminal amino acid sequence analysesto identify S-RNases associated with gametophytic self-incompatibility(SI) in this plant species. RNase activities corresponding toS_a and S_b, two of the four S-alleles tested, were identifiedby IEF and RNase activity staining. The S_a-RNase band reactedwith the anti-S₄serum prepared from Japanese pear (Pyrus serotina);no reaction with the antiserum was observed with the s_bRNaseband. When the s_a-RNase band was excised from an IEF gel stainedfor RNase activity, subjected to SDS-PAGE, and detected by immunoblotting,it appeared that this band consisted of a single protein thatreacted with the anti-s₄serum with M_r of about 28 kDa. With2D-PAGE and silver staining of the stylar extracts, all fourS-proteins could be successfully distinguished from each otherin the highly basic zone of the gel. Although S_b-, S_c-, andS_dproteins had roughly the same M_r of about 30 kDa, the S_c-proteinseemed to be slightly smaller than the S_b-protein and slightlylarger than the S_aprotein. In 2D-PAGE profiles as well, theS_a-protein had M_r of about 28 kDa, apparently smaller than theother three proteins. A bud sport, in which one of the two S-allelesof the original cultivar is impaired, was visualized as a lossof S_cprotein, which is consistent with the previous pollinationstudy. All four S-proteins reacted with the anti-S₄serum, probablybecause of the differing conformations of these S-proteins inthe IEF and 2D-PAGE gels. The S_a-protein in 2D-PAGE appearedto be identical to S_a-RNase in IEF; both bad the same M_r andwere reactive with the anti-S₄-serum. N-terminal amino acidsequence analysis of the four 5-proteins revealed that theywere highly homologous to each other and similar to the 5-RNasesof Malus, Pyrus, Scrophulariaceae, and Solanaceae. Taken together,RNases in the style are strongly suggested to be associatedwith the gametophytic SI of al- mond. This is the first reportidentofiying and characterizing S-RNase in almond. (Received July 11, 1996; Accepted December 26, 1996)     

Biosynthesis of GA73 Methyl Ester in Lygodium Ferns

Biosynthesis of GA73 methyl ester (GA73-Me), the principal antheridiogen in Lygodium ferns, was investigated. From the methanol extract of prothallia of Lygodium circinnatum, GA25, GA73, GA73-Me, GA88-Me, and a few unknown GA73 derivatives were detected by GC-MS. Because the presence of GA25 suggests that GA24, a direct precursor of GA25, could also be present in L. circinnatum prothallia, we used feeding experiments to investigate the possibility that GA24 is a precursor of GA73-Me. In L. circinnatum prothallia, [2H2]GA24 was converted into [2H2]GA73-Me and a trace amount of [2H2]GA9-Me, whereas [2H3]GA9 was converted into [2H3]GA9-Me and [2H3]monohydroxy-GA9-Me. Because GA73-Me, GA9-Me, and their monohydroxy derivatives had been identified by GC-MS from the culture medium of L. circinnatum prothallia, our results suggest that GA73-Me is biosynthesized from GA24 via GA73, and that neither GA9 nor GA9-Me is a precursor of GA73-Me. Though the possibility had been suggested that GA73-Me is biosynthesized from 9,15-cyclo-GA9 (GA103), [2H2]GA103 was not converted into [2H2]GA73-Me.     

High-performance liquid chromatography-thermospray mass spectrometry of 5,6-dihydroxyeicosatrienoate-1,5-lactone from tissue homogenates

We have developed a method for the analysis of 5,6-dihydroxyeicosatrienoate-1,5-lactone (5,6-DiHETriE-δ-lactone) in tissue homogenates, supplemented with NADPH and arachidonic acid [20:4(n−6)] as a substrate. During the incubation and the extraction, most of the 5,6-epoxyeicosatrienoic acid (5,6-EpETriE) was converted to 5,6-dihydroxyeicosatrienoic acid (5,6-DiHETriE), and most of the 5,6-DiHETriE was converted to 5,6-DiHETriE-δ-lactone. Consequently, the chief degradation product of 5,6-EpETriE and 5,6-DiHETriE in the incubation mixture was 5,6-DiHETriE-δ-lactone. 5,6-DiHETriE-δ-lactone, corresponding to [20:4(n−6)], was shown to be characterized by a high intensity of quasimolecular ions (MH⁺ and MNH₄⁺), using ion analysis obtained by reversed-phase HPLC-thermospray MS. On selected-ion monitoring (SIM) chromatograms of 5,6-DiHETriE-δ-lactone and with deuterium-labeled 15(s)-hydroxyeicosatetraenoic acid as the internal standard, the regression equation of the peak-area ratio and the amount of 5,6-DiHETriE-δ-lactone was y = 12.2x + 0.7 (r = 0.9996). 5,6-Epoxygenase activity was represented as the sum of the amount of 5,6-DiHETriE-δ-lactone, 5,6-EpETriE and 5,6-DiHETriE per mg protein, after 30 min in an incubation mixture. The activity from rat brain homogenate decreased considerably with growth of the rat.     

Cloning Vectors for Lactococci Based on a Plasmid Encoding Resistance to Cadmium

An 8.8-kb plasmid (pND302) was identified in Lactococcus lactis spp lactis M71 which encodes cadmium resistance (Cd^R). Most of the commercial lactococcal strains tested were sensitive to cadmium. Therefore, Cd^R should provide a useful selectable marker for constructing cloning vectors in lactococci. pND302 was mapped with a number of restriction enzymes and found to contain a unique EcoRI site suitable for cloning. Two E. coli/L. lactis shuttle cloning vectors, pND304 and pND624, were constructed by subcloning of the E. coli plasmids pBR322 and pGEM-7Zf(+) containing a 1.6-kb gene encoding nisin resistance (Nis^R) of lactococcal origin into the EcoRI site of pND302, separately. The E. coli DNA component of pND624 was removed and the resulting plasmid, pND625, consisted of only lactococcal DNA, expressing Nis^R and Cd^R, with two synthetic polylinkers that contain multiple restriction sites for versatile cloning. Both pND302 and pND625 can be transformed by electroporation into L. lactis LMO230 at 10³/μg DNA and maintained stably in LMO230. The results indicated that pND302 and pND625 are potential food-grade cloning vectors for lactococci. Received: 27 November 1995 / Accepted: 29 December 1995     

Inhibition of somatic embryo maturation in Sitka spruce [Picea sitchensis (Bong.) Carr.] by butylated hydroxytoluene,a volatile antioxidant released by parafilm

Head-space volatiles above embryogenicPicea sitchensis (Bong.) Carr. (Sitka spruce) tissues cultured in glass Petri dishes sealed with Parafilm M or cling-film, were captured on Tenax adsorption traps and analysed by gas chromatography / mass spectrometry. Each sealing system released a single major compound into the head-space; butylated hydroxytoluene from Parafilm M and 2-ethyl-1-hexanol from cling-film. After two weeks sealed under Parafilm M butylated hydroxytoluene accumulated to 1.1 g g^–1 FW in tissues and subsequent somatic embryo maturation was prevented. When butylated hydroxytoluene was supplied via the head-space (100 g/250 ml flask) 0.5 g g^–1 FW accumulated in tissues after two weeks and no somatic embryo maturation occurred. Potentially phytotoxic metabolites of butylated hydroxytoluene included a substituted stilbenequinone, butylated hydroxytoluene quinone methide and butylated hydroxytoluene dimer.     

Effects of Excitatory Amino Acids on Cerebral Oxygen Consumption and Blood Flow in Rat

This investigation tested the importance of excitatory amino acids' effects on regional cerebral O₂ consumption and the concomitant changes in cerebral blood flow (rCBF) in isoflurane anesthetized rats. In the glutamate or N-methyl-D-aspartate (NMDA) groups, 10^–2 M glutamate or NMDA was topically applied to the right cortex and the left cortex was used as a control. One mg/kg dizocilpine maleate (MK-801), a non-competitive NMDA receptor antagonist, was administered (iv) to the MK-801 group and saline was given to the control group. Cortical rCBF was determined using ¹⁴C-iodoantipyrine and regional O₂ extraction was measured microspectrophotometrically. Cerebral O₂ consumption increased 77% after glutamate (contralateral cortex: 9.0 ± 1.1 ml O₂/min/100 g, glutamate treated cortex: 15.9 ± 3.9), while a 46% increase was observed with the same concentration of NMDA (contralateral cortex: 9.8 ± 2.0, NMDA treated cortex: 14.3 ± 5.5). After MK-801, the O₂ consumption decreased to 37% of the control value (control cortex: 7.0 ± 1.3, MK-801 treated cortex: 2.6 ± 3.9). MK-801 significantly decreased cerebral O₂ extraction from 7.1 ± 1.3 ml O₂/100 ml (control cortex) to 5.3 ± 0.6 (MK-801 treated cortex). However, there was no significant difference in cerebral O₂ extraction between treated and contralateral cortex in either the glutamate or NMDA groups. The increase in O₂ consumption caused by glutamate or NMDA was coupled with increased rCBF. Glutamate increased rCBF from 95 ± 5 ml/min/100 g (contralateral cortex) to 165 ± 31 (treated cortex), while NMDA increased rCBF from 114 ± 12 (contralateral cortex) to 178 ± 60 (treated cortex). MK-801 decreased O₂ consumption with a lesser decrease of rCBF. The rCBF was 48 ± 9 in the MK-801 treated cortex and 99 ± 22 in the control cortex. Some substances produced by the activation of NMDA receptors may be related to the coupling of cerebral metabolism and blood flow, since after blockade of NMDA receptors with MK-801, this relationship is uncoupled. These findings suggest that glutamatergic processes have a major effect on cerebral O₂ consumption and that this is at least partly due to NMDA receptors.     

Effect of breast self-examination techniques on the risk of death from breast cancer

OBJECTIVE: To measure the effect of breast self-examination (BSE) technique and frequency on the risk of death from breast cancer. DESIGN: Case-control study nested within the Canadian National Breast Screening Study (NBSS). SETTING: The Canadian NBSS, a multicentre randomized controlled trial of screening for breast cancer in Canadian women. SUBJECTS: The case subjects were 163 women who had died from breast cancer and 57 women with distant metastases. Ten control subjects matched by 5-year age group, screening centre, year of enrolment and random allocation group were randomly selected for each case subject. EXPOSURE MEASURES: Self-reported BSE frequency before enrolment in the NBSS, annual self-reports of BSE frequency during the program and annual objective assessments of BSE technique. OUTCOME MEASURES: Odds ratios (ORs) associated with BSE practice were estimated by conditional multiple logistic regression modelling, which permitted control of covariates. RESULTS: Relative to women who, when assessed 2 years before diagnosis, examined their breasts visually, used their finger pads for palpation and examined with their 3 middle fingers, the OR for death from breast cancer or distant metastatic disease for women who omitted 1, 2 or 3 of these components was 2.20 (95% confidence interval [CI] 1.30 to 3.71, p = 0.003). The OR for women who omitted 1 of the 3 components was 1.82 (95% CI 1.00 to 3.29, p = 0.05), for those who omitted 2 of the 3 components, 2.84 (95% CI 1.44 to 5.59, p = 0.003), and for those who omitted all 3 components, 2.95 (95% CI 1.19 to 7.30, p = 0.02). The results remained unchanged after adjustment for potential confounders. CONCLUSION: The results, obtained with the use of prospectively collected data, suggest that the performance of specific BSE components may reduce the risk of death from breast cancer.