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 .     

C4 polymorphism in the dog: Molecular heterogeneity of the C4α and C4γ subunit chains

Using an immunoblotting technique and goat antihuman C4, we observed five distinct electrophoretic variants of C4 in a panel of 60 random dogs. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of immunoprecipitated C4 showed that dog C4 is composed of three polypeptide subunit chains (, , and ) and that structural variability occurs within the - and -chain regions. Two distinct molecular weight forms of both the C4- ( _A and _B) and C4-( _A and _B) chain were detected. The variant forms of C4 and C4 were found in association with particular C4 allotypes.     

Pathways of lymphocyte migration within the periarterial lymphoid sheath of rat spleen

Summary Male Wistar rats were injected intravenously with 5-(³H)uridine-labeled lymphocytes isolated from lymph nodes of syngeneic donors and enriched in T cells. After short periods of time (3 to 120 min after injection), labeled lymphocytes were localized in spleen compartments using autoradiography to identify routes of lymphocyte movement from blood into splenic parenchyma and to follow migration pathways of recirculating lymphocytes within the periarterial lymphoid sheath (PALS). Topographical analysis of labeled lymphocytes was performed in specific planes of PALS characterized by the diameter of the arterial vessel and termed PALS large, PALS medium, and PALS small (PALS L, PALS M, PALS S, respectively). Attention was also paid to accumulations of labeled lymphocytes close to the arterial vessel wall. Initially, labeled lymphocytes were localized in PALS S and PALS M near the terminal branching of arterial vessels and in the marginal zone (MZ). We conclude that lymphocytes emigrate from blood into splenic parenchyma within two white pulp compartments: in MZ, and directly within PALS through the wall of capillary vessels. The sequential accumulation of labeled cells near arterial vessels of increasing diameter suggests that the recirculating pool of lymphocytes migrates into the central part of PALS L by two routes: from MZ, and along arterial vessels from PALS S and PALS M.R.B. was a fellow of the Alexander von Humboldt-Stiftung, on leave from the Department of Histology and Embryology, Institut of Biostructure, Academy of Medicine, ul. Swiecickiego 6, PL-60-781 Pozna, Poland.     

Analysis of the regulation of adenosine 5′-phosphosulfate sulfotransferase activity inLemna minor L. using15N-density labeling

The role of de novo synthesis in the regulation of adenosine 5-phosphosulfate sulfotransferase activity by H₂S inLemna minor L. was investigate using density labeling with¹⁵N applied as¹⁵NO ₃ ^– in the culture medium. While adenosine 5-phosphosulfate sulfotransferase activity was rapidly reduced by H₂S and rapidly recovered upon removal of H₂S, O-acetyl-L-serine sulfhydrylase (EC 4.2.99.8) did not show changes in extractable activity in response to H₂S and could therefore be used as an internal marker enzyme for density labeling. The incorporation of¹⁵N into adenosine 5-phosphosulfate sulfotransferase was strongly reduced upon transfer of plants into a H₂S-containing atmosphere. Half-maximal labeling was reached only after 70–80 h compared to 40–50 h in the control. After removal of H₂S, adenosine 5-phosphosulfate sulfotransferase activity increased to the initial level within 20 h, and the enzyme reached halfmaximal labeling after only 15 h. The time course of the density increase of O-acetyl-L-serine sulfhydrylase was not affected very significantly by H₂S. These results provide evidence that de novo synthesis of enzyme protein is involved in the regulation of adenosine 5-phosphosulfate sulfotransferase activity by H₂S.Abbreviations APS adenosine 5-phosphosulfate - APSSTase adenosine 5-phosphosulfate sulfotransferase - BSA Bovine serum albumine - DTE dithioerythritol - OAS O-acetyl-L-serine - OASSase O-acetyl-L-serine sulfhydrylase - POPOP 1,4-bis-(5-phenyl-2-oxazolyl)-benzene - PPO 2,5-diphenyloxazole This is no. 9 in the series Regulation of Sulfate Assimilation in Plants     

The fine structure of the neural lobe of the mouse after transplantation under the kidney capsule

Summary The neurohypophysis of donor mice was implanted under the renal capsule of the recipients. The pituicytes survived while the neurosecretory axons disappeared. The ultrastructure of the glial cells was observed seven and nine weeks after transplantation. There were no signs of phagocytotic activity although remnants of axons were still present at seven weeks. The numerous processes of the pituicytes form a network with intercellular spaces wide in younger and narrower in older implants. The cells are connected by desmosomes and gap junctions. Pituicytes as well as blood vessels preserve their organotypic appearance. The transplant thus represents an experimental model for investigations on pituicytes in vivo in the absence of neurosecretory axons.Fellow of the Alexander von Humboldt-Stiftung. On leave from Department of Histology and Embryology, Institute of Biostructure, Academy of Medicine, Pozna, Poland     

Assessment of interleukin 1 and interleukin 2 effects on cycling and noncycling murine thymocytes

When mouse thymocytes are stimulated with PHA, the proliferative response is very low, unless the culture medium is enriched with interleukin 1 (IL-1)- or interleukin 2 (IL-2)-containing supernatants. Cytofluorometric analyses show, however, that PHA stimulation generates a significant number of cells with increased RNA content (transition from the G₀ to G₁ phase of the cell cycle). If IL-2 is added to such cultures, the activated cells complete their process of RNA synthesis and then enter the S phase. The use of IL-2-containing culture medium thus permits one to obtain a high correlation between the number of g₁ cells and [³H]thymidine incorporation (r = 0.97). Enrichment with IL-1-containing supernatants also results in a statistically significant correlation (r = 0.68), but the regression lines are markedly different for the two interleukins (s = 20.3 for IL-2 and s = 9.2 for IL-1), when analyzed after 48 hr of incubation. These observations suggest that the G₁ phase must be divided into two subcompartments, G_1a and G_1b, the G_1a-G_1b transition being an IL-2-dependent event. If the number of G_1b cells is used to establish correlations with [³H]thymidine incorporation, all values fall on the same regression line, regardless of culture conditions and of the addition of interleukins. It is concluded that IL-2 regulates lymphocyte proliferation at the level of RNA synthesis (G_1a-G_1b transition) rather than that of DNA synthesis (G₁-S transition).     

Characterization of a variant of carcinoembryonic antigen (CEA) using monoclonal and polyclonal anti-CEA antibodies

A variant of the carcinoembryonic antigen (CEA) with lower molecular weight than a CEA reference preparation has been separated from CEA. Using a polyclonal, spleen absorbed anti-CEA antiserum, the variant crossreacts with reference CEA in immunodiffusion. The CEA-activity of the variant has been demonstrated using an enzyme-immunoassay with monoclonal CEA specific antibodies. There is sufficient immunological evidence that this variant is a distinct antigen different from the crossreactive antigens described so far. The reactivity of the polyclonal anti-CEA antiserum with the CEA variant was abolished by absorption against the immobilized variant.     

Light dependence of protoplasmic streaming in Nitella flexilis L. as measured by means of laser-velocimetry

Laser-velocimetry was applied in order to study the effect of light on the velocity of protoplasmic streaming (pps) in Characean cells. A change from dark to light (= 6 W · m^–2) leads to an acceleration of streaming by about 15–30% with a time-constant of approx. 300 s. The transition from light to dark causes a transient decrease of velocity below the original dark level. This response occurs with a time constant of about 500 s. It returns to its initial value with a time-constant of about 2000 s. This may indicate that a control loop of cytosolic homeostasis takes a decrease in pCa more seriously than an increase. A possible involvement of temperature effects caused by illumination was excluded by measuring the influence of temperature. Steady-state velocity of streaming changed by 5% per 1° C. Irradiation with infra-red light ( > 780 nm) did not cause a change in velocity. The absence of a light effect on streaming velocity in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) shows that photosynthesis and not phytochrome is involved. The role of light-induced changes of pCa is discussed, especially with respect to the hypothesis of Vanselow and Hansen (1989, J. Membr. Biol. 110, 175–187) that photosynthesis acts on the plasmalemma K⁺-channel via light-induced uptake of Ca²⁺ into the chloroplasts.Abbreviations and Symbols ASF auto structure function - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - pps protoplasmic streaming - _L, _D, _C time-constants of the light and dark responses, and of a putative Ca-control system Financial support by the Deutsche Forschungsgemeinschaft is gratefully acknowledged. The first author was granted a scholarship by the state of Schleswig-Holstein. We are indebted to Prof. Dr. G. Pfister for technical advice and helpful discussions and to Mrs. E. Götting for drawing the figures.