Verhaltensontogenese der Habitatwahl beim Teichrohrs?nger (Acrocephalus scirpaceus)

Zusammenfassung In Anpassung an seinen aus vertikalen Vegetationsstrukturen zusammengesetzten Lebensraum besitzt der Teichrohrsänger die beste morphologische Ausstattung aller sechs mitteleuropäischer Rohrsängerarten für Vertikalklettern. An jungen Teichrohrsängern wurde überprüft, ob frühkindliche Erfahrungen auf verschiedenen Sitzstangen (auf senkrechten=K_s, senkrechten und waagerechten=K_m, waagerechten=V_w und waagerechten Sitzstangen mit Futter belohnt=V_w+) die spätere Wahl dieser Strukturelemente beeinflussen. Die lokomotorische Aktivität der vier Aufzuchtsgruppen wurde im Zweifachwahlversuch (horizontale gegen vertikale Sitzstangen) getestet. Mit Ausnahme der auf senkrechten Sitzstangen aufgezogenen Vögel (K_s), deren Wahlverhalten gleichverteilt war (Abb. 2), bevorzugten alle anderen Gruppen das vertikale Testsubstrat (Abb. 2 und 3). Auch die Belohnung mit Futter als positiver Verstärker der Horizontalelemente (V_w+) machte diese nur wenig attraktiver (Tab. 1). Alle Gruppen nutzten während der Testperiode das vertikale Substrat zunehmend stärker (Tab. 1). Die Zunahme rührte bei den Vögeln der beiden Kontrollgruppen (K_s und K_m) und der Versuchsgruppe waagerecht (V_w) alleine von der Erfahrung in der Versuchssituation her (Tab. 2). Die Nutzung vertikaler bzw. horizontaler Strukturelemente durch junge Teichrohrsänger wird somit bestimmt durch: 1. eine angeborene Präferenz für das artgemäße Substrat, 2. einen Novitätseffekt, bedingt durch die Aufzuchterfahrung (Wahlverhalten der K_s-Gruppe, Abb. 2), 3. Eigenerfahrung bei der Nutzung verschiedener Substrate im Versuch. Zusätzliche Wahlversuche später im Jahr (Oktober bis Dezember) mit denselben Versuchsvögeln zeigten keine Änderungen in der Substratwahl (Abb. 4).

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Ontogeny of habitat choice in the Reed Warbler (Acrocephalus scirpaceus)

Summary The Reed Warbler shows the most specialized morphological traits for vertical climbing among the six central EuropeanAcrocephalus species. We consider this as an adaptation to the vertical structures in its habitat. In experiments with young Reed Warblers I tested whether early experience with different perches has an influence on the choice of these structures later on in life. Locomotory activity of the following groups was tested in double choice experiments (horizontal versus vertical perches):Control group vertical (K_s): raised on vertical perches, Control group mixed habitat (K_m): raised on vertical and horizontal perches, Test group horizontal (V_w): raised on horizontal perches, Test group horizontal, plus rewards (V_w+): raised on horizontal perches and additional feeders attached to the bars. With the exception of Control group vertical (K_s) which shows no choice preference (Fig. 2) all other birds prefered the vertical test substrate (Fig. 2 and 3). Even the reward (food) as a positive reinforcement of horizontal elements had little effect on their attractiveness (Tab. 1). An increase in the choice of the vertical substrate could be observed throughout the 3-day test period in all groups (Tab. 1). This increase was only due to the experience in the test situation for the birds in both control groups (K_s and K_m) and the Test group horizontal (V_w) (Tab. 2). Therefore the use of vertical or horizontal structures in Reed Warblers is determind by: 1. An innate preference for species-specific substrate, 2. Novelty due to early experience (choice behaviour of Control group vertical, Fig. 2), 3. Experience while using different substrates (experience in the test situation) which optimizes substrate choice according to proprioceptive learning. Additional choice experiments with the same test birds later on in the year (October to December) revealed the same results. Therefore it seems unlikely that there exists a preprogrammed change in substrate choice in the course of a year (Fig. 4). Although choice experiments of this kind can only include a limited part of the habitat-scheme, they are useful experimental designs for investigating specialized species whose habitats can be simulated closely in the laboratory.

     

2-Heptanone and 10-hydroxy-trans-dec-2-enoic acid in the mandibular glands of worker honey bees of different ages

Summary The contents of 2-heptanone and 10-hydroxy-trans-dec-2-enoic acid present in the mandibular glands of worker bees of various ages were determined by gas chromatography.The content of 2-heptanone was dependent on the physiological development rather than the chronological age of the bee. It remained at zero for hive bees and reached a maximum of ca. 40 40 g/bee after the bees began to forage.The content of 10-hydroxy-trans-dec-2-enoic acid found in worker bee heads increased with age and/or in connection with normal changes in the type of social activity of the bee. Only trace amounts of this acid were found in newly emerged bees, but up to 60 g/bee were found after about 3 weeks of age.

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Zusammenfassung 2-Heptanon und 10-Hydroxy-trans-2-Decensäure, die in den Mandibeldrüsen verschiedenalter Arbeiterbienen vorkommen, wurden gas-chromatographisch mengenmäßig bestimmt.Das Vorkommen von 2-Heptanon hing von der physiologischen Entwicklung, nicht aber direkt vom Alter der Bienen ab. Stockbienen enthielten kein Heptanon, Flugbienen hatten bis zu ca. 40 g.10-Hydroxy-trans-2-Decensäure nimmt mit dem Alter der Bienen oder auch im Zusammenhang mit dem normalen Wechsel von einer sozialen Tätigkeit zur anderen zu. Bei neugeschlüpften Bienen fanden sich nur Spuren, aber nach 3 Wochen fanden sich bis zu 60 g pro Biene.

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Dedicated to Prof. von Frisch on the occasion of his 80th birthday.     

Purification of bovine colostrumβ-galactosideα(2–6)sialyltransferase to near homogeneity by affinity chromatography

Cytidine-5-monophospho-N-acetylneuraminic acid:-galactoside 2-6sialyltransferase was purified from bovine colostrum by two sequential affinity chromatography steps on CDP-ethanolamine-Sepharose and CDP-ethanolamine-(N-caproylamino-)-Sepharose, respectively. While the conditions for elution were those of Paulsonet al. [J Biol Chem (1977) 252:3256–62], the ligand of the second affinity column was coupled to Sepharose by using 6-aminocaproic acid as linker. The ease of this procedure allows rapid synthesis of bulk quantities of ligand.Highly purified preparations of sialyltransferase were obtained which moved on gradient gel electrophoresis as a single band of 76 kDa and on dodecylsulphate electrophoresis as a single band of 54 kDa. The product of the reaction between lactose and CMP-N-acetylneuraminic acid catalyzed by the purified sialyltransferase was identified by high-resolution 500 MHz¹H-NMR spectroscopy as Neu5Ac2-6Gal1-4Glc.     

Photon yield of O2 evolution and chlorophyll fluorescence characteristics at 77 K among vascular plants of diverse origins

Photon yields of oxygen evolution at saturating CO₂ were determined for 44 species of vascular plants, representing widely diverse taxa, habitats, life forms and growth conditions. The photonyield values on the basis of absorbed light ( ^a) were remarkably constant among plants possessing the same pathway of photosynthetic CO₂ fixation, provided the plants had not been subjected to environmental stress. The mean ^a value ±SE for 37 C₃ species was 0.106±0.001 O₂·photon^-1. The five C₄ species exhibited lower photon yields and greater variation than the C₃ species ( ^a=0.0692±0.004). The ^a values for the two Crassulaceanacid-metabolism species were similar to those of C₃ species. Leaf chlorophyll content had little influence on ^a over the range found in normal, healthy leaves. Chlorophyll fluorescence characteristics at 77 K were determined for the same leaves as used for the photon-yield measurements. Considerable variation in fluorescence emission both at 692 nm and at 734 nm, was found 1) among the different species; 2) between the upper and lower surfaces of the same leaves; and 3) between sun and shade leaves of the same species. By contrast, the ratio of variable to maximum fluorescence emission at 692 nm (F_v/F_M, 692) remained remarkably constant (The mean value for the C₃ species was 0.832±0.004). High-light treatments of shade leaves resulted in a reduction in both ^a and the F_v/F_M, 692 ratio. The extent of the reductions increased with time of exposure to bright light. A linear relationship was obtained when ^a was plotted against F_v/F_M, 692. The results show that determinations of the photon yield of O₂ evolution and the F_v/F_M, 692 ratio can serve as excellent quantitative measures of photoinhibition of overall photosynthetic energy-conversion system and of photochemistry of photosystem II, respectively. This is especially valuable in field work where it is often impossible to obtain appropriate controls.Abbreviations and symbols CAM Crassulacean acid metabolism - PFD photon flux density (photon fluence rate) - PSI, PSII photosystem I, II - F_o, F_M, F_v instantaneous, maximum, variable fluorescence emission - absorptance - ^a photon yield (absorbed light) - ⁱ photon yield (incident light) C.I.W.-D.P.B. Publication No. 923     

Initiation and growth characteristics of suspension cultures of Calendula officinalis cells

Callus cultures of marigold (Calendula officinalis L.) were induced on Murashige and Skoog medium with different concentrations of auxin (dichlorophenoxyacetic acid (2,4-D) or indole-3-acetic acid (IAA) and cytokinin (kinetin or 6-(,-dimethylallylamino)purine (2iP). Of all hormone combinations used in the medium, two were the most efficient in promoting callus development: 1.81 M (0.4 mg l^–1) 2,4-D and 1.85 M (0.4 mg l^–1) kinetin (0.4d–0.4k culture) or 0.45 M (0.1 mg l^–1) 2,4-D and 2.02 M (0.5 mg l^–1) 2iP (0.1d–0.5p culture). These combinations were selected to induce cell suspension cultures. The suspension cultures were maintained under light or dark conditions. The light stimulated cell aggregation in the cultures. In both cultures cells were undifferentiated under darkness, whereas in the light, rhyzogenesis was observed in 0.1d–0.5p culture. The cell growth and protein and oleanolic acid contents were determined. Initially, biomass production was similar under light and dark conditions, but after 7–8 months from the induction the cell growth was reduced by approximately 30% in the light, whereas the cell growth of the cultures maintained under darkness did not reveal any changes. The presence of oleanolic acid was detected in the suspension cultures kept in darkness. This compound reached two quantitative peaks: in the lag and stationary phases –- beyond the active growth phase of the culture cycle and its concentration was several times higher in 0.1d–0.5p culture than that in 0.4d–0.4k culture. It was for the first time that callus and suspension cultures were induced from the marigold plant.     

Immunofixation for C2 typing: C2 allotypes in Spaniards in relation to HLA,Bf and C4

Summary C2 typing is performed by immunofixation with anti-C2 antiserum instead of by a hemolytic overlay. This method gives sharp band definition, is less cumbersome than the hemolytic overlay, gel files are easily made, and it also enables one to describe putative new nonhemolytic variants. C2 allele frequencies were studied in a sample of the normal Spanish population and were found to be similar to other Caucasoids. HLA-Bw62,-Cw3, and-DR4 were significantly associated with C2 B. Concordantly, the only C2^*B extended HLA haplotype found in family material was Bw62-Cw3-Bw6-(DR4)-Bf^*S-C2^*B-C4A^*3 B^*2-(GLO^*1). C4A^*4 B^*2 and C4A^*4 B^*4 are not found within the same haplotype together with C2^*B and Bw62 or Bw22 respectively, nor do other C2^*B haplotypes occur with common HLA-B alleles. These results may favour the hypothesis that the Bw62-C2^*B haplotype is produced by one mutation arising in the Bw62-C2^*C haplotype and that subsequent crossovers can explain other C2^*B haplotypes (including Bw22-C2^*B).     

A simple,single-tube radioisotopic assay for the phosphorylation/inactivation activity of the pyruvate,orthophosphate dikinase regulatory protein

A simple, single-tube radiolsotopic method has been developed to assay the relative phosphorylation (inaetivation) activity of the bifunctional regulatory protein (RP) of C₄-leaf pyruvate,orthophosphate dikinase (PPDK) in desalted leaf homogenates and partially purified preparations. RP catalyzes the inactivation of maize PPDK by phosphorylation of Thr-456, utilizing [-P]ADP as the specific phosphoryl donor. Existing spectrophotometric and radioisotopic assays for the detection of RP activity are either relatively insensitive or labor-intensive and timeconsuming. We describe a modified radioisotopic assay that couples the synthesis of [-³²P]ADP by exogenous adenylate kinase with the subsequent RP-catalyzed [-³²P]ADP-dependent phosphorylation of exogenous maize PPDK. The incorporation of [-³²P] is dependent on the initial concentrations of ATP and PPDK, as well as the presence of active RP. Desalted leaf homogenates of C₃ species fail to catalyze ³²P incorporation into exogenous maize PPDK. Conversely, heterologous systems containing the maize target enzyme and leaf homogenats of other C₄ species result in PPDK-specific ³²P-incorporation. This simple radioisotopic assay is at least 40-times more sensitive than the routine spectrophotometric assay, and qualitatively exhibits comparable sensitivity and requires significantly less time than the currently available radioisotopic RP assay. The present assay reliably generates [-³²P]ADP and as such may be useful for studies of other systems requiring -labeled ADP, which is not commercially available.Abbrevlations Ap₅A P¹, P⁵-di(adenosine-5)-pentaphosphate - Bicine N,N-bis[2-hydroxyethyl]glycine - DTT dithiothreitol - PEI poly(ethyleneimine) - PEP phosphoenolpyruvate - PEPC PEP carboxylase (E.C.4.1.1.31) - PPDK pyruvate,orthophosphate dikinase (E.C.2.7.9.1) - RP PPDK regulatory protein     

Interchange-trisomics in barley

Observations on morphology, sterility and cytology of some interchangetrisomics obtained from the progeny of radiation induced interchangeheterozygotes in a 6-rowed barley (Hordeum vulgare L.) var. K₁₂ are described. The different interchange trisomics show distinct phenotypic expression and have been classified as bushy, slender, robust, semi-erect and pseudonormal on the basis of their gross morphology, leaf characteristics, awn and spike lengths. All interchange trisomics show pollen and ovule sterility which varies between plants. Meiotic behaviour is described. The five different interchange trisomics appear to have different extra chromosomes.     

Interactions of γ-L-glutamyltaurine with excitatory aminoacidergic neurotransmission

The in vitro effects of -L-glutamyltaurine on different stages of excitatory aminoacidergic neurotransmission were tested with -D-glutamyltaurine as reference. -L-Glutamyltaurine enhanced the K⁺-stimulated release of [³H]glutamate from cerebral cortical slices (25% at 0.1 mM) and slightly inhibited the uptake by crude brain synaptosomal preparations (about 10% at 1 mM). -L-Glutamyltaurine was also a weak displacer of glutamate and its agonists from their binding sites in brain synaptic membrane preparations, being, however, less selective to quisqualate (QA) sites than -D-glutamyltaurine. The basal influx of Ca²⁺ into cultured cerebellar granular cells was not affected by 1 mM -L-glutamyltaurine, but the glutamate- and its agonist-activated influx was significantly inhibited in low-Mg²⁺ (0.1 mM) and Mg²⁺-free media. The glutamate-evoked increase in free intracellular Ca²⁺ and the kainate-activated formation of cGMP in cerebellar slices were both markedly inhibited by 0.1 mM -L-giutamyltaurine. We propose that -L-glutamyltaurine may act as endogenous modulator in excitatory aminoacidergic neurotransmission.     

Formation of the imidazolides of dinucleotides under potentially prebiotic conditions

Summary Imidazolides of dinucleotides such as ImpApA can be formed from the corresponding dinucleotides in a two-stage process, which gives up to 15% yields under potentially prebiotic conditions. First a solution of the dinucleotide and sodium trimetaphosphate is dried out at constant temperature and humidity. This produces polyphosphates such as pnApA in excellent yield (80%). The products are dissolved in water, imidazole is added, and the solution is dried out again. This yields the 5-phosphorimidazolides.Abbreviations P₃! trimetaphosphate - A adenosine - U uridine - EDTA ethylenediaminetetraacetic acid - Ap adenosine 2(3)-phosphate - Ap! adenosine cyclic 2:3-phosphate - pA adenosine 5-phosphate - pA²p adenosine 2, 5-diphosphate - pA³p adenosine 3, 5-diphosphate - pAp! 5-phospho-adenosine cyclic 2:3-phosphate - ATP adenosine 5-triphosphate - ImpA adenosine 5-phosphorimidazolide - A²pA adenylyl-[25]-adenosine - A³pA adenylyl-[35]-adenosine - A²pU adenylyl-[25]-uridine - A³pU adenylyl-[35]-uridine - pA²pA 5-phosphoadenylyl-[25]-adenosine - pA³pA 5-phospho-adenylyl-[35]-adenosine - pA²pU 5-phospho-adenylyl-[25]-uridine - pA³pU 5-phospho-adenylyl-[35]-uridine - pApN (N= A, U) 5-phosphate of a dinucleoside phosphate - p_nApN (N = A, U; n = 2, 3, 4.) 5-polyphosphate of a dinucleoside phosphate - ImpA²pA imidazolide of pA²pA - ImpA³pA imidazolide of pA³pA - ImpA²pU imidazolide of pA²pU - ImpA³pU imidazolide of pA³pU - ImpApN imidazolide of pApN     

Role of mitogen-activated protein kinase pathway in reactive oxygen species-mediated endothelin-1-induced β-myosin heavy chain gene expression and cardiomyocyte hypertrophy

Endothelin-1 (ET-1) has been found to increase cardiac -myosin heavy chain (-MyHC) gene expression and induce hypertrophy in cardiomyocytes. ET-1 has been demonstrated to increase intracellular reactive oxygen species (ROS) in cardiomyocytes. The exact molecular mechanism by which ROS regulate ET-1-induced -MyHC gene expression and hypertrophy in cardiomyocytes, however, has not yet been fully described. We aim to elucidate the molecular regulatory mechanism of ROS on ET-1-induced -MyHC gene expression and hypertrophic signaling in neonatal rat cardiomyocytes. Following stimulation with ET-1, cultured neonatal rat cardiomyocytes were examined for ³H-leucine incorporation and -MyHC promoter activities. The effects of antioxidant pretreatment on ET-1-induced cardiac hypertrophy and mitogen-activated protein kinase (MAPKs) phosphorylation were studied to elucidate the redox-sensitive pathway in cardiomyocyte hypertrophy and -MyHC gene expression. ET-1 increased ³H-leucine incorporation and -MyHC promoter activities, which were blocked by the specific ET_A receptor antagonist BQ-485. Antioxidants significantly reduced ET-1-induced ³H-leucine incorporation, -MyHC gene promoter activities and MAPK (extracellular signal-regulated kinase, p38, and c-Jun NH₂ -terminal kinase) phosphorylation. Both PD98059 and SB203580 inhibited ET-1-increased ³H-leucine incorporation and -MyHC promoter activities. Co-transfection of the dominant negative mutant of Ras, Raf, and MEK1 decreased the ET-1-induced -MyHC promoter activities, suggesting that the Ras-Raf-MAPK pathway is required for ET-1 action. Truncation analysis of the -MyHC gene promoter showed that the activator protein-2 (AP-2)/specificity protein-1 (SP-1) binding site(s) were(was) important cis-element(s) in ET-1-induced -MyHC gene expression. Moreover, ET-1-induced AP-2 and SP-1 binding activities were also inhibited by antioxidant. These data demonstrate the involvement of ROS in ET-1-induced hypertrophic responses and -MyHC expression. ROS mediate ET-1-induced activation of MAPK pathways, which culminates in hypertrophic responses and -MyHC expression. Tzu-Hurng Cheng, Neng-Lang Shih: These authors have equally contributed to this work     

H2: heterodisulfide oxidoreductase,a second energy-conserving system in the methanogenic strain Gö1

Washed everted vesicles of the methanogenic bacterium strain Gö1 catalyzed an H₂-dependent reduction of the heterodisulfide of HS-CoM (2-mercaptoethanesulfonate) and HS-HTP (7-mercaptoheptanoylthreonine phosphate) (CoM-S-S-HTP). This process was independent of coenzyme F₄₂₀ and was coupled to proton translocation across the cytoplasmic membrane into the lumen of the everted vesicles. The maximal H⁺/CoM-S-S-HTP ratio was 2. The tranmembrane electrochemical gradient thereby generated was shown to induce ATP synthesis from ADP+P_i, exhibiting a stoichiometry of 1 ATP synthesized per 2 CoM-S-S-HTP reduced (H⁺/ATP=4). ATP formation was inhibited by the uncoupler 3,5-di-tert-butyl-4-hydroxy-benzylidene-malononitrile (SF 6847) and by the ATP synthase inhibitor N,N-dicyclohexylcarbodiimide (DCCD). This energy-conserving system showed a stringent coupling. The addition of HS-CoM and HS-HTP at 1 mM each decreased the heterodisulfide reductase activity to 50% of the control. Membranes from Methanolobus tindarius showed F₄₂₀H₂-dependent but no H₂-dependent heterodisulfide oxidoreductase activity. Neither of these activities was detectable in membranes of Methanococcus thermolithotrophicus.Abbreviations _H+ transmembrane electrochemical gradient of H⁺ - CoM-SH 2-mercaptoethanesulfonate - F₄₂₀ (N-l-lactyl--l-glutamyl)-l-glutamic acid phosphodiester of 7,8-didemethyl-8-hydroxy-5-deazariboflavin-5-phosphate - F₄₂₀H₂ reduced F₄₂₀ - HTP-SH 7-mercaptoheptanoylthreonine phosphate - DCCD N,N-dicyclohexylcarbodiimide - SF 6847 3,5-di-ert-butyl-4-hydroxybenzylidenemalononitrile - Mb. Methanobacterium - Ml. Methanolobus - Mc. Methanococcus - MV methylviologen - BV benzylviologen - MTZ metronidazole     

Significance of rooting depth in mire plants: Evidence from natural <Superscript>15</Superscript>N abundance

Variation in stable nitrogen isotope ratios (¹⁵N) was assessed for plants comprising two wetland communities, a bog-fen system and a flood plain, in central Japan. ¹⁵N of 12 species from the bog-fen system and six species from the flood plain were remarkably variable, ranging from –5.9 to +1.1 and from +3.1 to +8.7, respectively. Phragmites australis exhibited the highest ¹⁵N value at both sites. Rooting depth also differed greatly with plant species, ranging from 5cm to over 200cm in the bog-fen system. There was a tendency for plants having deeper root systems to exhibit higher ¹⁵N values; plant ¹⁵N was positively associated with rooting depth. Moreover, an increasing gradient of peat ¹⁵N was found along with depth. This evidence, together with the fact that inorganic nitrogen was depleted under a deep-rooted Phragmites australis stand, strongly suggests that deep-rooted plants actually absorb nitrogen from the deep peat layer. Thus, we successfully demonstrated the diverse traits of nitrogen nutrition among mire plants using stable isotope analysis. The ecological significance of deep rooting in mire plants is that it enables those plants to monopolize nutrients in deep substratum layers. This advantage should compensate for any consequential structural and/or physiological costs. Good evidence of the benefits of deep rooting is provided by the fact that Phragmites australis dominates as a tall mire grass.     

Kinetics of transient pump currents generated by the (H,K)-ATPase after an ATP concentration jump

Summary (H,K)-ATPase containing membranes from hog stomach were attached to black lipid membranes. Currents induced by an ATP concentration jump were recorded and analyzed. A sum of three exponentials ( ₁ ^-1 400 sec^–1, ₂ ^-1 100 sec^–1, ₃ ^-1 10 sec^–1; T = 300 K, pH 6, MgCl₂ 3 mm, no K⁺) was fitted to the transient signal. The dependence of the resulting time constants and the peak current on electrolyte composition, ATP conversion rate, temperature, and membrane conductivity was recorded. The results are consistent with a reaction scheme similar to that proposed by Albers and Post for the NaK-ATPase. Based on this model the following assignments were made: ₂ corresponds to ATP binding and exchange with caged ATP. ₁ describes the phosphorylation reaction E₁ · ATP E₁P. The third, slowest time constant ₃ is tentatively assigned to the E₁P E₂P transition. This is the first electrogenic step and is accelerated at high pH and by ATP via a low affinity binding site. The second electrogenic step is the transition from E₂K to E₁H. The E₂K E₁H equilibrium is influenced by potassium with an apparent K _0.5 of 3 mm and by the pH. Low pH and low potassium concentration stabilize the E₁ conformation.The authors wish to thank Dr. E. Grell and Mr. G. Schimmack. MPI Frankfurt, for synthesizing caged ATP, Mrs. S. Meister, Hoechst AG Frankfurt, for valuable help to prepare the (H,K)-ATPase, and Dr. W. Haase, MPI Frankfurt, for electron microscope pictures. (H,K)-ATPase for preliminary experiments was provided by Dr. W. Beil, Medizinische Hochschule Hannover, Dr. H. Swarts, University of Nijmegen, and Dr. G. Metzger, Hoechst AG Frankfurt. The work was supported by the Deutsche Forschungsgemeinschaft (SFB 169).     

Fluorescence microscopy and radiolabeling of C3 and C4 chloroplasts using diisothiocyanatostilbene disulfonic acid as a marker for the phosphate translocator

The usefulness of 4,4-diisothiocyanatostilbene-2,2-disulfonic acid (DIDS) for in-situ studies of the chloroplast phosphate translocator was evaluated by fluorescence microscopy and radiolabeling of spinach (Spinacia oleracea L.) (C₃ plant) and maize (Zea mays L.) (C₄ plant) chloroplasts. In maize mesophyll and bundle-sheath chloroplasts and in spinach chloroplasts that were either intact, broken or swollen, DIDS fluorescence was only associated with the chloroplast envelope. Intact chloroplasts often had fluorescent patches corresponding to concave regions of the chloroplast which we assume to be regions enriched in DIDS-binding sites.Incubation of intact or broken spinach chloroplasts or maize mesophyll chloroplasts with [³H₂]DIDS resulted in the labeling of a single polypeptide (relative molecular mass, M_r, 30 kDa) in the envelope fraction, in each case. Label in the stromal fraction was not detected when intact chloroplasts were incubated with [³H₂]DIDS. However, when broken chloroplasts were incubated with [³H₂]DIDS, several polypeptides of various molecular masses were labeled, but not the 30×31-kDa polypeptide. In thylakoid fractions from both broken and intact chloroplasts, a single 30×31-kDa polypeptide was labeled inconsistently. When a mixture of intact maize mesophyll and bundle-sheath chloroplasts was labeled with [³H₂]DIDS, extracts of whole chloroplasts displayed radioactivity only in the 30×31-kDa band.We conclude that DIDS is a valuable probe for the in-situ identification and characterization of the 30-kDa protein — the presumptive phosphate translocator — in C₃ and C₄ chloroplasts since DIDS (1) does not penetrate the inner membrane of the envelope of intact chloroplasts and, therefore, (2) does not bind internal sites in intact chloroplasts, and (3) only binds the 30-kDa protein in the inner membrane of the envelope.Abbreviations CBB Coomassie brilliant blue - DIC differential interference contrast optics - DIDS 4,4-diisothiocyanatostilbene-2,2-disulfonic acid - [³H₂]DIDS 1,2-ditritio-1,2-(2,2-disulfo-4,4-diisothiocyano)diphenylethane - kDa kilodalton - M_r relative molecular mass - PGA 3-phosphoglycerate - Pitranslocator phosphate translocator - SDS sodium dodecyl sulfate     

Effect of orchard and postharvest application of daminozide on ethylene synthesis by apple fruit

The effects of daminozide (butanedioic acid-2,2-dimethylhydrazide) on ethylene synthesis by apple fruits were investigated. The objective was to determine the effects of postharvest applications as compared to the standard application of diaminozide in the orchard. Immersion in a solution containing 4.25 g L^–1 active ingredient for 5 min delayed the rise in ethylene production in individual Cox apples at 15°C by about 2 days, whereas orchard application of 0.85 g L^–1 caused delays of about 3 days. Both modes of application depressed the maximal rate of ethylene production attained by ripe apples by about 30%. Daminozide did not affect the stimulation of respiration by ethylene treatment of Gloster apples, but it delayed the increase in ethylene synthesis. Daminozide applied immediately after harvest delayed the rise in ethylene synthesis in Golden Delicious held at 15°C, but it was less effective when applied 48 h after harvest or when apples were held at 5°C. Exposure to 1–2 l L^–1 ethylene for 48 h was less effective in promoting the rise in ethylene in daminozidetreated Cox and Gloster apples than in untreated fruit. High (100–1000 l L^–1) concentrations of ethylene more or less overcame the daminozide effect. Apples absorbed about 40% of surface-applied [¹⁴C]daminozide in 48 h, but more than 90% of the radioactivity in the fruit was recovered from the peel and outer 1 cm of the cortex. Daminozide was partly converted to carbon dioxide and other metabolites.     

Polymorphism of the HLA DR1 haplotype in the Israeli population investigated at the serological,cellular, and genomic levels

In the present report, we used serological, cellular, and restriction fragment length polymorphism (RFLP) to investigate the DR1 haplotype in the Israeli population. We describe an Israeli homozygous typing cell (HTC), HLA-DwLVA, which defines a new lymphocyte-activating determinant associated with Bw65, DR1 and distinct from Dwl. The parents of this donor, non-Ashkenazi Algerian Jews, are first cousins and share HLA-Cw8, Bw65, BfS, DR1, DQw1, DPw4. No specificity could be assigned to HLA-DwLVA using the 91 Ninth Workshop HTCs. Two families and forty unrelated DR1 individuals were studied with DwLVA and a panel of DR1/Dw1 HTCs. HLA-DwLVA showed segregation as a single determinant within families. This new specificity was present in 24 out of 40 (60%) unrelated DR1 individuals, indicating that in the Israeli population DwLVA is the main lymphocyte-defined determinant associated with the serologically defined DRI specificity, in contrast to non-Jewish Caucasoids where DR1 is significantly associated with Dw1. The vast majority of DwLVA-positive carriers were also Bw65 carriers, indicating that Bw65, DR1, DwLVA may represent a typical allele combination in the Israeli population. The RFLP analysis established the correlation of certain RFLPs with Dw1 and DwLVA. In addition, we describe a cluster of RFLPs that may correspond to a new Dw subtype associated with DR1, for which no serological and cellular reagents have been described so far.     

Structure-function studies of mEGF: Probing the type Iβ-turn between residues 25 and 26

The interaction between epidermal growth factor (EGF) and its receptor molecule is not completely understood and has received much attention recently. Studies combining site-directed mutagenesis and NMR spectroscopy have identified a number of EGF residues that are required for activity and are believed to interact directly with the receptor. Instead of focusing on these residues, this study combines site-directed mutagenesis and NMR spectroscopy to probe the role of the type I-bend located between residues 25 and 26 of the N-terminal subdomain of the protein. Ser25 of murine EGF is replaced by Pro in an attempt to stabilize this turn conformation to produce a variant of mEGF with increased activity relative to that for the native protein. Ser25 is also replaced by Ala, which is found at position 25 in human EGF (hEGF), as a more conservative replacement. Receptor binding studies demonstrate that both mutations produce about a 30% reduction in binding affinity, which is shown to result from local changes within the loop or minor perturbations of residues neighboring the loop rather than from long-range perturbations of the-sheet of the N-terminal subdomain. The type I-turn appears to remain intact in both mutants; however, replacement with Pro seems to introduce more flexibility into this region of the protein. These results demonstrate that perturbation of this-turn has little effect on EGF-receptor interactions.Abbreviations EGF epidermal growth factor - h human - m murine - TGF type transforming growth factor - NMR nuclear magnetic resonance - [S2A]mEGF mEGF missing the N-terminal asparagine and with the serine at position 2 replaced by alanine - [S2A,S25A]mEGF and [S2A, S25P]mEGF replacement of serine at position 25 in [S2A]mEGF by alanine and proline, respectively - ¹²⁵I-mEGF ¹²⁵I-labeled mEGF - DMEM Dulbecco's modified Eagle's medium - FCS fetal calf serum - HEPES N-(2-hydroxyethyl)piperazine-N-2-ethanesulfonic acid - BSA bovine serum albumin - COSY correlated spectroscopy - DQCOSY double-quantum filtered COSY - NOESY nuclear Overhauser spectroscopy - NOE nuclear Overhauser effect - TOCSY total correlation spectroscopy - ³ J(H-H^N) vicinal spin-spin coupling constant between amide proton and -proton - DSS 2,2-dimethyl-2-silapentane-5-sulfonate - chemical shift in ppm - ppm parts per million,     

Heterogeneous Long Chain Acyl-CoA Synthetases Control Distribution of Individual Fatty Acids in Newly-Formed Glycerolipids of Neuronal Cells Undergoing Neurite Outgrowth

Using PC12 cells undergoing neurite outgrowth, we studied the activation of various fatty acids, of different chain lengths and degrees of saturation, by long chain acyl-CoA synthetases (LCASs). Cells treated with nerve growth factor (NGF) were labeled with [³H]glycerol, [³H]oleic acid (OA) or [³H]arachidonic acid (AA) in the presence of other unlabeled fatty acids of endogenous or exogenous origin. Triacsin C (4.8 M), an inhibitor of acyl-CoA synthetase, decreased the incorporation of exogenous [³H]OA into glycerolipids by 30–90%, and increased by about 60% the accumulation of free [³H]OA in the cells. However it did not affect the incorporation of endogenous fatty acids nor of exogenous [³H]AA into phospholipids, suggesting that LCASs which activate exogenous AA and at least some endogenous fatty acids are relatively insensitive to this drug. Activities of the LCAS that is specific for AA (ACS), or of the non-specific LCAS which activates OA and other fatty acids (OCS), were much higher in microsomal and cytoplasmic fractions than in mitochondria or nuclei. The Vmax and Km values of ACS and OCS in microsomes were 12 and 0.7 nmol/min/mg protein and 70 and 37 M, respectively; and in cytoplasm, 6 and 0.6 nmol/ min/mg protein and 38 and 60 M, respectively. Triacsin C (2–33 M) did not affect ACS activity in microsomal or cytoplasmal fractions, but inhibited OCS activities dose-dependently and competitively: IC₅₀ and apparent Ki values were 13.5 M and 14 M in microsomes, and 3.8 M and 4 M in cytoplasm. NGF stimulated the activities of the LCASs, and, consistently, the incorporation of the various fatty acids into glycerolipids. These data indicate that LCASs are heterogeneous with respect to their intracellular locations, substrate specificities, kinetic characteristics and sensitivities to triacsin C; and that this heterogeneity affects the extents to which individual fatty acids are utilized to form glycerolipids.     

Analysis of the donor-specific cytotoxic T lymphocyte repertoire in a patient with a long term surviving allograft. Frequency, specificity, and phenotype of donor-reactive T cell receptor (TCR)-alpha beta+ and TCR-gamma delta+ clones

In the present study the transplant specific CTL repertoire of a patient (HLA:A1,3, B8,18, Cw5,7 DR3, DQw2, DPw3) with a long term surviving HLA mismatched kidney graft (HLA: A1,24 B8,27 Cw2,7, DR3, w13 DQw2,6 DPw1,3) has been investigated. This patient was unable to generate specific cytolytic activity against donor-derived PHA-blasts in the MLC in which donor spleen cells or B lymphoblastoid cell line were used as stimulator cells. In addition, the CTL precursor frequencies against donor alloantigens were very low (1/67,000). The patient had otherwise normal immune responses in vivo and in vitro and no signs of transplant rejection. Transplant specific CTL clones were generated in high frequencies (1/195) from T cell bulk cultures activated by PHA in the absence of any sensitization by donor Ag in vitro. The repertoire of 14 donor-reactive CTL clones (12 TCR-alpha beta+ and 2 TCR-gamma delta+) was analyzed. Two TCR-alpha beta+ CD8+ clones were specific for B27. Ten TCR-alpha beta+ CTL clones directed against class II HLA Ag were isolated. Seven of these were CD4+ and recognized DRw13 (3), DQw6 (3), and DPw1 (1), whereas three of these clones were CD4-CD8+ recognizing DRw13 (1) and DQw6 (2). In addition, two donor-specific TCR-gamma delta+ CTL clones were obtained recognizing HLA-A9(23,24) and DQw6. Our data indicate that the precursors of CTL clones specifically directed against donor class I or II HLA Ag are not deleted from the repertoire and that part of this reactivity resides in the TCR-gamma delta+ fraction.