Enhanced quantitative resistance against fungal disease by combinatorial expression of different barley antifungal proteins in transgenic tobacco

cDNAs encoding three proteins from barley ( Hordeum vulgare ), a class-II chitinase (CHI), a class-II β-1,3-glucanase (GLU) and a Type-I ribosome-inactivating protein (RIP) were expressed in tobacco plants under the control of the CaMV 35S-promoter. High-level expression of the transferred genes was detected in the transgenic plants by Northern and Western blot analysis. The leader peptides in CHI and GLU led to accumulation of these proteins in the intercellular space of tobacco leaves. RIP, which is naturally deposited in the cytosol of barley endosperm cells, was expressed either in its original cytosolic form or fused to a plant secretion peptide (spRIP). Fungal infection assays revealed that expression of the individual genes in each case resulted in an increased protection against the soilborne fungal pathogen Rhizoctonia solani , which infects a range of plant species including tobacco. To create a situation similar to 'multi-gene' tolerance, which traditional breeding experience has shown to provide crops with a longer-lasting protection, several of these antifungal genes were combined and protection against fungal attack resulting from their co-expression in planta was evaluated. Transgenic tobacco lines were generated with tandemly arranged genes coding for RIP and CHI as well as GLU and CHI. The performance of tobacco plants co-expressing the barley transgenes GLU/ CHI or CHI/RIP in a Rhizoctonia solani infection assay revealed significantly enhanced protection against fungal attack when compared with the protection levels obtained with corresponding isogenic lines expressing a single barley transgene to a similar level. The data indicate synergistic protective interaction of the co-expressed anti-fungal proteins in vivo .     

Response to Gravity by Zea mays Seedlings : I. Time Course of the Response

Gravistimulation induces an asymmetric distribution of free indole-3-acetic acid (IAA) in the cortex-epidermis of the Zea mays L. cv `Stowells Evergreen' mesocotyl within 15 minutes, the shortest time tested. IAA was measured by an isotope dilution method as the pentaflurobenzyl ester. The per cent IAA in the lower half of the mesocotyl cortex was 56 to 57% at 15, 30, and 90 minutes after stimulus initiation. Curvature is detectable in the mesocotyl within 3 minutes after beginning gravitropic stimulation. The rate of curvature of the mesocotyl increases during the first 60 minutes to a maximum of about 30° per hour. Thus, the growth asymmetry continues to increase for 45 minutes after hormone asymmetry is established.

Free IAA occurs predominantly in the stele of the mesocotyl whereas esterified IAA is mainly in the mesocotyl cortex-epidermis. This compartmentation may permit determining in which tissue the hormone asymmetry arises. Current data suggest the asymmetry originated in the stele.

     

Continuous measurement of minute ventilation and gaseous metabolism of newborn infants

A system of instrumentation for continuous measurement of gaseous metabolism and minute volume (VI) in the human newborn is described. O2 uptake and CO2 production are measured by open-circuit techniques utilizing a Servomex OA184 differential paramagnetic O2 analyzer and a BEckman LB-2 infrared CO2 analyzer. VI is measured with bias-flow pneumotachometry. Bench performance is described, methodological errors are defined, and clinical data are presented. The instrumentation is capable of safe, accurate, and continuous measurement of respiratory and metabolic variables in low-birth-weight infants.     

Ecophysiological investigations on lichens of the Negev desert

Summary Previous publications have reported on investigations of CO₂ exchange in the desert lichenRamalina maciformis both in its natural habitat in the Negev and in the laboratory. Utilizing laboratory data, net photosynthesis and dark respiration were expressed as mathematical functions of the most important environmental factors. Based on these relationships, a model is developed that allows one to predict CO₂ exchange of the plant. Input data are light intensity, temperature, and water content of the thallus, together with a measure of the rate of the seasonal change of photosynthetic and respiratory activity. The validity of the model is tested by comparing simulated daily courses of CO₂ uptake and release of the lichen with independent results of CO₂ exchange measurements conducted in the field during and after the condensation of dew. The sensitivity of the model is shown by simulating changes in the input data of temperature and water content of the lichen.This paper is dedicated to Dr. h.c. Oscar Klement on the occasion of his 80th birthday     

Functional Morphology of the Olfactory Organ in Spinachia spinachia (L.) (Teleostei,Gasterosteidae)

The functional morphology of the olfactory organ in Spinachia spinachia (L.), which has only a single nare, was studied by light microscopy, scanning electron microscopy, and experimental investigations. It was shown that only the incoming water passes over the olfactory epithelium. The device for ventilating this olfactory organ is an accessory ventilation sac activated by respiratory pressure changes in the buccal cavity. This one-way water current over the olfactory epithelium in a monotrematous olfactory organ was found to be possible because of the morphology of the olfactory organ combined with movements of the lateral wall of the olfactory organ and the nasal tube during respiration. The olfactory epithelium is divided into irregular islets. Both ciliated receptor cells and microvillous receptor cells are present.     

Inactivation and stabilization of stabilisins in neat organic solvents

The stability of the serine proteases from Bacillus amyloliquefaciens (subtillisin BPN') and Bacillus licheniformis (subtilisin Carlsberg) was investigated in various anhydrous solvents at 45 degrees C. The half-life of subtilisin BPN' in dimethyl-formamide dramatically depends on the pH of the aqueous solutions from which the enzyme was lyophilized, increasing from 48 min to 20 h when the pH is raised from 6.0 to 7.9. Both subtilisins exhibited substantial inactivation during multihour incubations in tert-amyl alcohol and acetonitrile when enzymatic activities were also measured in these solvents; however, when the enzymes were assayed in water instead, hardly any loss of activity was detected. This surprising difference appears to stem from the partitioning of the bound water essential for catalytic activity from the enzymes into the solvents. When assayed in organic solvents, this time-dependent stripping of water results in decay of enzymatic activity; however, when assayed in water, where the dehydrated subtilisins can undergo rehydration thereby recovering catalytic activity, little inactivation is observed. In agreement with this hypothesis, the addition of small quantities of water tert-amyl alcohol stabilized the subtilisins in it even when enzymatic activity was measured in the nonaqueous solvent. Ester substrates (vinyl butyrate and trichloroethyl butyrate) greatly enhanced the stability of both subtilisins in organic solvents possibly because of the formation of the acyl-enzymes.     

Incorporation of phosphonic acid diesters into lipid model membranes. Part II. X-ray and neutron diffraction studies.

Mixtures of egg phosphatidylcholine and phosphonic acid diethyl or dibutyl esters of the general type RP(O)(OR')2 with R = hexane or dodecane were studied at room temperature in the fluid lamellar state by X-ray and by neutron diffraction. Generally a molar ratio of lipid and ester of 1:0.5 was used. Additionally an equimolar lipid/ester mixture of hexane phosphonic acid diethyl ester was studied. Depending on the ester used and its concentration a single L alpha-phase was observed above a certain water content which changes to an L alpha + water two phase system at high water concentration. Despite the large amounts of the amphiphilic ester molecules incorporated in the membrane and their high molecular asymmetry, the mixtures qualitatively show the typical hydration and swelling behaviour of non-charged lipid membranes. However, the incorporation of the esters induces a higher hydration capacity, a lateral extension and a decrease in membrane thickness. The position of the ester molecules and their orientation in the membrane were determined by neutron diffraction using partially deuterated esters. The esters were found to be located with their phosphonic moiety near or in the lipid/water interface. The lamellar structure contradicts this location of the cone-shaped ester molecules which should increase the tendency to form hexagonal structures. However, the experimental findings can be understood if one considers a partial interdigitation of the last hydrocarbon groups of the lipid chains accompanied by a larger disorder in the hydrophobic centre of the membrane. In the case of hexane phosphonic acid dibutyl ester, a vertical translocation of the ester takes place below a certain water content where it is distributed between two locations at the lipid water interface and the centre of the membrane.     

Establishment of a germ-line competent C57BL/6 embryonic stem cell line

Embryonic stem (ES) cell lines have been derived from blastocysts of the inbred mouse strain C57BL/6. The highest frequencies of ES cell colonies were observed when blastocysts were explanted directly onto growth-arrested feeder layers of 5637 human bladder carcinoma cells in the presence of conditioned medium. One of the male ES cell lines tested (BL/6-III) was shown to be karyotypically stable and germ-line competent when introduced into BALB/c host blastocysts. These results demonstrate that ES cell lines from inbred mouse strains other than 129/Sv may be used as vectors to introduce selected mutations into the germ-line of mice.