Developmental regulation of expression of the malate synthase gene in transgenic plants

The cucumber malate synthase (MS) gene, including 1856 bp of 5 non-trnascribed sequence, has been transferred into Petunia (Mitchell) and Nicotiana plumbaginifolia plants using an Agrobacterium binary vector. The transferred gene is found in variable copy number in different transformants, and is stably transmitted in each case as a single Mendelian character. Transgene mRNA accumulates in the seedling during the first three days of germination, then declines in amount as the cotyledons emerge from the seed. The decline is more pronounced in light-grown seedlings than in dark-grown seedlings. Expression of the MS transgene is also detected at a low level in petals of transformed Petunia plants. In these respects the pattern of MS gene expression is similar in cucumber and in trnasformed plants, showing that the transferred DNA fragment contains a functional MS gene. A 1076 bp fragment of 5 sequence was linked to the -glucuronidase reporter gene and transferred into Nicotiana, where it was shown to direct temporal and spatial patterns of expression similar to that of the complete MS gene. However, histochemical localisation of -glucuronidase activity demonstrated that the chimaeric gene is expressed not only in cotyledons of transgenic plants, but also in endosperm and some hypocotyl cells during early germination. The relevance of these findings to the control of malate synthase gene expression is discussed.     

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.     

Hemolysis of rabbit erythrocytes in the presence of copper ions

The hemolysis of red blood cells (RBC) induced by Cu(II) is modified by ceruloplasmin (Cp) and albumin. The time course of hemolysis for rabbit RBC by Cu(II) consisted of two parts, an induction period followed by a catastrophic lysis period. The induction period decreased and the lysis rate increased with increasing Cu(II) concentration. Cp or albumin, modified Cu(II) induced hemolysis, by increasing the duration of the induction period and decreasing the overall rate of hemolysis of RBC. The catastrophic lysis period coincided with a sharp increase in the formation of metHb within the cell and in a rapid uptake of Cu(II). The presence of Cp led to an increase in the induction period prior to the rapid increase in metHb formation and in Cu(II) uptake. Porcine Cp was prepared with either two or three nonprosthetic copper binding sites (sites where Cu(II) is easily removed by passing over Chelex-100). Cp with three nonprosthetic binding sites gave more protection than Cp with two. Likewise, albumin can be prepared with three and five nonprosthetic copper binding sites. The albumin with five sites gave more protection than the albumin with three sites.     

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.     

Direct analysis of root zone data in a microculture system

Disks were isolated from young leaves of winter rape plants and grown in vitro at ambient (15°C) or low (2°C) temperatures for two weeks. In the control disks the growth cessation and beginning of chlorophyll degradation were observed after 1 week of culture. In the low-temperature treated disks the expansion of cells was slower than that in the control material but it continued for two weeks and was accompanied by a marked accumulation of dry matter. Practically, no chlorophyll degradation was observed. The low temperature treatment brought about the decrease in the frost killing temperature of the tissue which was associated with its increased capacity to subcool water. A short (18 h) exposure of the cold-grown leaf disks to slight frost (–5°C) increased further their resistance to freezing, despite the fact that the subcooling capacity of disks decreased in result of the treatment. Therefore, the two stages of hardening, observed previously for the whole plants, can also be detected in the isolated material. In the cold-grown disks, a transient accumulation of reducing sugars but a steady decrease in ATP and water-soluble protein contents were observed. These observations indicate that tissue isolation might affect processes involved in the functional adaptation of cells to cold.Abbreviations DTA differential thermal analysis - T_k50 frost killing temperature - T_in ice nucleation temperature     

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.     

Retrovirus-mediated insertional mutagenesis: phagemid rescue of flanking DNA by selecting plasmid ori

A method for screening recombinant lambda libraries was devised to select phage containing genomic regions containing provirus insertions of retroviruses that carry the kanamycin and G418 resistance factor neo and the origin of replication derived from pBR322 (oripBR). Such recombinants are phagemids, able to replicate as bacteriophages or as plasmids under lambda repressor control. lambda repressor was cloned into a plasmid derived from pSC101 that is compatible with pBR322-derived phagemids. A strain carrying this plasmid may be used to select phagemids derived from a single proviral insertion with 100% efficiency from complex recombinant libraries. Homologous recombination between proviral long terminal repeats was observed at a rate of 10(-4)/plaque-forming unit in recABC+ strains. Despite this frequency, intact phagemids are easily recovered as phage after temperature shift to 42 degrees C. Since oripBR itself is a selectable marker in this system, the method could be applied to recover any sequence carrying the ori sequence from pBR322.     

Analysis of the topological changes induced on cells exposed to adhesive or mechanical stimuli

Fluorescent probes are widely used to study cell structure and function. However, few reports were devoted to a quantitative analysis of the intracellular distribution of fluorescent markers. In the present work, we describe the topographical changes of surface and cytoskeletal markers on individual cells subjected to adhesive or mechanical interaction. Conjugates were prepared with a cytotoxic T-lymphocyte clone and target cells. Specific antigens, membrane phospholipids, surface glycoconjugates, and polymerized actin were labeled with fluorescent antibodies or biochemical probes. The analysis of fluorescence distributions in conjugates demonstrated a selective reorganization of the plasma membrane with a gathering of some molecular species in the intercellular adhesion area. Furthermore, individual phagocytic cells were sucked into glass micropipets, then stained with fluorescent phallacidin to analyze the effect of mechanical efforts on the cytoskeleton organization. The concentration of polymerized actin was found to be similar in mechanicallyinduced protrusions and whole cells. It is concluded that adhesive interactions may result in marked cell polarization and formation of membrane zones with a particular biochemical composition. The submembranar cytoskeleton might play a role in this process.     

Floristic and structural variation in the Tamaulipan thornscrub,northeastern Mexico

Abstract. A generalized research strategy is presented for identifying the ecological effects of the physical environment and management in a poorly known region of subtropical, semiarid thornscrub in northeastern Mexico. Vegetation samples were stratified across a small number of climatic subregions, substrate types and topographic situations. Classification analysis and PCo A of the species x site matrix of incidence data after application of the Information Statistic were used. The analyses suggested that the regional variation in climate, substrates and topography was responsible for the major floristic differences in the vegetation. The distributions of most plant species were related to the variation in the physical environment. PCo A of the species x site cover data after application of the Bray-Curtis dissimilarity metric revealed evidence of vegetation change due to overgrazing in each major floristic group, but not to selective cutting for timber and firewood.     

Isotopic Fractionation of Hydrogen in Plants

The naturally-occurring stable isotopes deuterium and hydrogen are fractionated by a number of physical and biological processes. Deuterium has a tendency to precipitate out first from a moist air mass. Thus ground water will become isotopically lighter with an increase in latitude, altitude, or distance inland. Water taken up by the plant from the soil undergoes little change until evapotranspiration results in leaf water becoming isotopically heavier. Thus hydrogen isotopes in plants can reveal something of geography (groundwater) and climate. Hydrogen isotopes undergo little fractionation by passage through the food chain, although plant parasites tend to be enriched in D as compared to their hosts, possibly due to higher rates of transpiration in the parasitic plants. The splitting of water in photosynthesis results in the lighter isotope being incorporated into organic matter. An even larger isotopic fractionation results during lipid synthesis and other processes involving the pyruvate dehydrogenase complex. Differences in metabolic pathway between species can be detected by D/H ratios. Hydrogen isotopic differences can be detected between CAM, C₄, and C₃ species. Within C₄ plants, the NADP-ME plants are isotopically distinguishable from NAD-ME and PEP-CK plants.