Age determination of Antarctic krill using fluorescence and image analysis of size

Summary The ages of 252 mature female krill, collected from Prydz Bay in January 1985, were determined using length frequency analysis and the fluorescent age pigment (FAP) technique. Results of both methods suggest 6 years classes for adult krill. Correspondence between the ages determined by the two techniques is generally within one year. The animals were also analyzed by a computerized image analysis system, which recorded a large suite of size and shape parameters. The accuracy of discriminant functions constructed to relate the image analysis parameters to age approached 90% for the ages defined by length frequency, and 52% for physiological age.     

Effects of wounds on the terpene content of twigs of maritime pine (Pinus pinaster Ait.)

Summary The effect of wounds on the volatile terpene composition of the living bark of Pinus pinaster Ait. (maritime pine) twigs was investigated with respect to the processes of mono- and sesquiterpene hydrocarbon biosynthesis. The large increase in the amounts of - and -pinene is a characteristic feature after a mechanical injury, whereas the quantities of the other terpenes are only slightly increased. This is due to the reactivation of the resin duct secretory cells of primary origin located in cortical tissues. The effect of wounding is observed over a long period and the terpene profiles are very different at the end of the experiments as compared with the initial profiles of the same tissues. The traumatic essential oil (obtained after mechanical traumatism) resembles an oleoresin extracted from tissues of secondary origin. Statistical analysis underlines the effects of the between-tree variations and of the dates of application of the wound.     

Physical mapping of the von Recklinghausen neurofibromatosis region on chromosome 17

The von Recklinghausen neurofibromatosis (NF1) locus has been linked to chromosome 17, and recent linkage analyses place the gene on the proximal long arm. NF1 probably resides in 17q11.2, since two unrelated NF1 patients have been identified who possess constitutional reciprocal translocations involving 17q11.2 with chromosomes 1 and 22. We have used a somatic-cell hybrid from the t(17;22) individual, along with other hybrid cell lines, to order probes around the NF1 locus. An additional probe, 17L1, has been isolated from a NotI linking library made from flow-sorted chromosome 17 material and has been mapped to a region immediately proximal to the translocation breakpoint. While neither NF1 translocation breakpoint has yet been identified by pulse-field gel analysis, an overlap between two probes, EW206 and EW207, has been detected. Furthermore, we have identified the breakpoint in a non-NF1 translocation, SP-3, on the proximal side of the NF1 locus. This breakpoint has been helpful in creating a 1,000-kb pulsed-field map, which includes the closely linked NF1 probes HHH202 and TH17.19. The combined somatic-cell hybrid and pulsed-field gel analysis we report here favors the probe order D17Z1-HHH202-TH17.19-CRYB1-17L1-NF1- (EW206, EW207, EW203, L581, L946)-(ERBB2, ERBA1). The agreement in probe ordering between linkage analysis and physical mapping is excellent, and the availability of translocation breakpoints in NF1 should now greatly assist the cloning of this locus.     

Electroporation of Bradyrhizobium japonicum

Summary Electroporation offers a fast, efficient and reproducible way to introduce DNA into bacteria. We have successfully used this technique to transform two commercially important strains of Bradyrhizobium japonicum, the nitrogen-fixing soybean symbiont. Initially, electroporation conditions were optimized using plasmid DNA which had been prepared from the same B. japonicum strain into which the{imDNA was to b}e transformed. Efficiencies of 10⁵-10⁶ transformants/g DNA were obtained for strains USDA 110 and 61A152 with ready-to-use frozen cells. Successful electroporation of B. japonicum with plasmid DNA prepared from Escherichia coli varied with the E. coli strain from which the plasmid was purified. The highest transformation efficiencies (10⁴ transformants/g DNA) were obtained using DNA prepared from a dcm ^– dam ^– strain of E. coli. This suggests that routine isolation of DNA from an E. coli strain incapable of DNA modification should help in increasing transformation efficiencies for other strains of bacteria where DNA restriction appears to be a significant obstacle to successful transformation. We have also monitored the rate of spontaneous mutation in electroporated cells and saw no significant difference in the frequency of streptomycin resistance for electroporated cells compared to control cells.     

Studies on the biosynthesis and metabolism of the phytoalexin lubimin and related compounds in Datura stramonium L.

Arachidonic acid, cellulase, CuSO₄, a sonicate of Phytophthora infestans mycelium and a spore suspension of Penicillium chrysogenum all elicited the formation of the sesquiterpenoid phytoalexins lubimin, 3-hydroxylubimin and rishitin in fruit cavities of Datura stramonium. 3-Hydroxylubimin was the predominant phytoalexin formed after treatment of the fruits with arachidonic acid, cellulase and the P. infestans preparation. Copper sulphate was a potent elicitor of lubimin but not 3-hydroxylubimin. The fungus P. chrysogenum metabolized lubimin and 3-hydroxylubimin to 15-dihydrolubimin and 3-hydroxy-15-dihydrolubimin respectively, both in fruit cavities inoculated with spores of this fungus and in pure culture. The 15-dihydrolubimin formed in the fruits by the fungus was further metabolized (by the fruits) to both isolubimin and 3-hydroxy-15-dihydrolubimin. The precursor-product relationships between all of the subject compounds was investigated by feeding experiments with ³H-labelled compounds. 2-Dehydro-[15-³H₁]lubimin was rapidly and efficiently incorporated into lubimin and may be the direct precursor of lubimin in planta. 3-Hydroxy[2-³H₁]lubimin was incorporated into the nor-eudesmane rishitin but 10-epi-3-hydroxy[2-³H₁]lubimin was not. An updated scheme for the biosynthesis and metabolism of lubimin and related compounds in infected tissues of solanaceous plants is presented.We thank Mr Vic Swetez for the provision of plant material, Mrs Margaret Huffee for technical assistance, Dr David Ewing for help with obtaining NMR spectra, and the Agricultural and Food Research Council for financial support.     

Short-term changes in carbon-isotope discrimination identify transitions between C3 and C4 carboxylation during Crassulacean acid metabolism

Short-term measurements of instantaneous carbon-isotope discrimination have been determined from mass-spectrometric analyses of CO₂ collected online during gas exchange for the epiphytic bromeliad Tillandsia utriculata L. Using this technique, the isotopic signature of CO₂ exchange for each phase of Crassulacean acid metabolism (CAM) has been characterised. During night-time fixation of CO₂ (Phase I), discrimination () ranged from 4.4 to 6.6, equivalent to an effective carbon-isotope ratio (¹³C) of –12.3 to –14.5 versus Pee Dee Belemnite (PDB). These values reflected the gross photosynthetic balance between net CO₂ uptake and refixation of respiratory CO₂, characteristic of CAM in the Bromeliaceae. When for the relative proportion of external (p _a ) and internal (p _i) CO₂ is taken into account, calculated p _i/p _a decreased during the later part of the dark period from 0.68 to 0.48. Measurements of during Phase II, early in the light period, showed the transition between C₄ and C₃ pathways, with carboxylation being increasingly dominated by ribulose bisphosphate carboxylase (Rubisco) as increased from 10.5 to 21.2 During decarboxylation in the light period (Phase III), CO₂ leaked out of the leaf and the inherent discrimination of Rubisco was expressed. The value of calculated from on-line measurements (64.4) showed that the CO₂ lost was considerably enriched in ¹³C, and this was confirmed by direct analysis of the CO₂ diffusing out into a CO₂-free atmosphere ( ¹³C = + 51.6 versus PDB). Instantaneous discrimination was characteristic of the C₃ pathway during Phase IV (late in the light period), but a reduction in showed an increasing contribution from phosphoenolpyruvate carboxylase. The results from this non-invasive technique confirm the observations that double carboxylation involving both phosphoenolpyruvate carboxylase and Rubisco occurs during the transient phases of CAM (II and IV) in the light period.Abbreviations and Symbols CAM Crassulacean acid metabolism - H⁺ (dawn-dusk) variation in titratable acidity - ¹³C carbonisotope ratio of plant organic material, relative to Pee Dee Belemnite (vs. PDB) - discrimination against ¹³CO₂, - p _i, p _a internal, external partial pressures of CO₂ - Rubisco ribulose1,5-bisphosphate carboxylase - PAR photosynthetically active radiation - PEPCase phosphoenolpyruvate carboxylase We are grateful for financial support in respect of research grants (GR3/5360, GR3/6419) and a studentship awarded by the Natural Environment Research Council, UK.     

Class I major histocompatibility complex cDNA clones from sheep thymus: alternative splicing could make a long cytoplasmic tail

To investigate the class I major histocompatibility complex (MHC) genes expressed in the young sheep thymus, a cDNA library was screened with a human HLA-B7 cDNA probe under conditions of relaxed stringency. Thirteen clones were isolated and found by partial sequences to fall into five classes, requiring the expression of at least three loci. One sequence was found six times, almost half of the total, and may thus represent the major message expressed in the young sheep thymus. One of the clones was found to have failed to excise the intron between cytoplasmic exons 7 and 8, leading to the predicted synthesis of a cytoplasmic domain 23 amino acids longer than the other sheep sequences, and 15 amino acids longer than any cytoplasmic domain previously described. The sequences of all the clones were found to be most similar to bovine, and least similar to mouse class I MHC sequences.The nucleotide sequence data reported in this paper have been sunmitted to the GenBank nucleotide sequence database and have been assigned the accession numbers M 34672-6.     

Genotype-specific response of cultured broccoli (Brassica oleracea var. italica) anthers to cytokinins

Anther culture medium was prepared with different types and concentrations of cytokinins to gain greater insight into the control of embryo formation during Brassica oleracea L. var. italica (broccoli) anther culture. The independent addition of the four cytokinins tested had widely divergent effects dependent upon cytokinin concentration and the genetic background of the test plants. All cytokinins were generally inhibitory at high concentrations, however, individual plants showed significant stimulation of embyro formation at typical physiological levels. The influence of cytokinins was highly cultivar-specific, some lines were stimulated, others inhibited and still other test lines were largely unaffected. Although the addition of cytokinins was needed for embryo formation for some plants, in no instance were cytokinins able to replace the inductive effect of high-temperature treatments.     

Nitrogen inputs to a marine embayment: the importance of groundwater

We examined the importance of nitrogen inputs from groundwater and runoff in a small coastal marine cove on Cape Cod, MA, USA. We evaluated groundwater inputs by three different methods: a water budget, assuming discharge equals recharge; direct measurements of discharge using bell jars; and a budget of water and salt at the mouth of the Cove over several tidal cycles. The lowest estimates were obtained by using a water budget and the highest estimates were obtained using a budget of water and salt at the Cove mouth. Overall there was more than a five fold difference in the freshwater inputs calculated by using these methods. Nitrogen in groundwater appears to be largely derived from on site septic systems. Average nitrate concentrations were highest in the region where building density was greatest. Nitrate in groundwater appeared to behave conservatively in sandy sediments where groundwater flow rates were high (> 11/m²/h), indicating that denitrification was not substantially reducing external nitrogen loading to the Cove. Nitrogen inputs from groundwater were approximately 300 mmol-N/m³/y of Cove water. Road runoff contributed an additional 60 mmol/m³/y. Total nitrogen inputs from groundwater and road runoff to this cove were similar in magnitude to river dominated estuaries in urbanized areas in the United States.     

Differential effects of lithium on agonist-stimulated inositol polyphosphate formation in rat cerebral cortex slices: Selective actions on muscarinic cholinergic responses

Recent data indicate that BMY 7378 demonstrates high affinity, selectivity and low intrinsic activity at hippocampal 5-HT_1A receptors, suggesting that BMY 7378 may represent the first selective 5-HT_1A functional antagonist. The present study examined the agonist and antagonist properties of BMY 7378 at spinal cord 5-HT_1A receptors. In electrophysiological studies, iontophoretic administration of either the 5-HT_1A agonist 8-OH-DPAT (43.8 ± 5.4 nA) or BMY 7378 (46.3 ± 5.2 nA) significantly inhibited the firing rate of wide-dynamic-range dorsal horn units indicating that BMY 7378 demonstrates significant intrinsic activity at spinal cord 5-HT_1A receptors. Concomitant BMY 7378 and 8-OH-DPAT administration identified no BMY 7378 ejection current (20–100 nA) which antagonized the 8-OH-DPAT induced inhibition of dorsal horn unit activity. In behavioral studies in the spinal rat, 8-OH-DPAT increased the animals' sensitivity to noxious levels of mechanical stimulation (ED₅₀ = 269 ± 24 nmol/kg) as did BMY 7378 (ED₅₀ = 295 ± 70 nmol/kg) with no statistically significant difference in the maximal response (Y_max) observed. Concomitant BMY 7378 and 8-OH-DPAT administration identified no BMY 7378 dose (10–1100 nmol/kg) which blocked the hyperalgesic effect of 8-OH-DPAT, rather, each drug produced similar effects which were additive. Further, the 5-HT_1A agonist effects of BMY 7378 were blocked by the 5-HT_1A antagonist, spiperone. Therefore, both the electrophysiologic and reflex data indicate that BMY 7378 possesses significant intrinsic activity at spinal cord 5-HT_1A receptors, and like 8-OH-DPAT is a partial agonist at these receptors. Differences in BMY 7378 intrinsic activity at spinal cord as opposed to hippocampal 5-HT_1A receptors are discussed in terms of regional differences in G-proteins coupled to 5-HT_1A receptors in these two CNS regions.