Volume 45, Issue 6

Plants defend themselves against pathogen attack by activating a whole set of defense responses, most of them relying on transcriptional activation of plant defense genes. The same responses are induced by treatment of plant cells with elicitors released from the pathogen or from the plant surface. Several plant/elicitor combinations have been used successfully as experimental systems to investigate the molecular basis of plant defense responses. Receptor-like structures on the plasma membrane of plant cells appear to bind the elicitors. Thereby, intracellular signal transduction chains are initiated which finally result in the activation of plant defense genes. A better understanding of the molecular mechanisms of early processes in plant defense responses, as provided by these studies, may in the long term help to develop environmentally safe plant protection methods for agriculture.

The major outer membrane protein of Rhodobacter capsulatus 37 b4 (capsule-free) was iso­lated. Strong porin-activity was observed after reconstitution into artificial lipid bilayer mem­branes with a single channel conductance of 3.15 nS in Im KC1. The porin migrated as a broad, single band (M r above 90,000) on sodium dodecyl sulfate polyacrylamide gel electro­ phoresis and dissociated into a single species of polypeptides (M r 36,000) on treatment with EDTA (10 mM at 30 °C, 20 min) or by heating (100 °C, 5 min). Analytical ultracentrifugation studies demonstrated the native porin to be a trimer. The monomers chromatofocused as a single, sharp peak on fast performance liquid chromatography and only one band, corre­ sponding to an isoelectric point of about 4.0, was obtained on isoelectric focusing. Gas-phase sequencing of the 23 N-terminal residues revealed Glu-Val-Lys-Leu-Ser-Gly-Asp-Ala-Arg-Met-Gly-Val-Met-Tyr-Asn-Gly-Asp-Asp-X-Asn-Phe-Ser-Ser.

From Rhytidiadelphus squarrosus the hitherto unknown biflavone 5′-hydroxyrobustaflavone and the three known biflavonoids 5′-hydroxyamentoflavone,5′,3′′′-dihydroxyamentoflavone and 2,3-dihydro-5′-hydroxyamentoflavone have been isolated.

Bud exudate of Populus deltoides clones originating from six central and eastern American states was examined by gas chromatography/mass spectrometry and the components were identified. The principal components of the bud exudate were the flavone galangin, the flavanone pinocembrin and the flavanonol pinobanksin, together with the related compounds pinocembrin chalcone, pinobanksin methyl ether and pinobanksin-3-acetate. The bud exudate composition was very different from that seen in a North American poplar of the section Tacamahaca, P. balsamifera. The bud exudates from all P. deltoides specimens were similar excepting that from Vermont.

Bud exudate of Populus balsamifera contains an array of characteristic compounds. Bud exudate of P. deltoides contains a different array of characteristic compounds. The exudate of P. x jackii contains compounds characteristic of both P. balsamifera and P. deltoides, indicating its hybrid origin.

The epicuticular waxes from the leaves of maples (Acer pseudoplatanus L.) contained hydrocarbons (6.9%), wax esters (5.5%), aldehydes (38.1%), primary alcohols (10.2%) and fatty acids (17.1%). In addition to these common wax lipids, benzyl acyl esters (2.1%) and triterpenoids were also present. β-Sitosterol, β-amyrin and 24-methylene-cycloartenol were found in the form of acetates (14.4%). β-Amyrin was also present as free alcohol (4.9%) and esterified with long chain fatty acids ( ~ 0.7%).

Unorganized callus and leaf/root-differentiating callus cultures of Pimpinella major have been established in liquid nutrient medium. Their capacity to accumulate rare phenylpropanoids such as epoxy-pseudoisoeugenol tiglate, epoxy-anol tiglate and anol tiglate was compared with that of seedlings and whole plants. The unorganized callus cultures were not able to accumulate any phenylpropanoids. In comparison, the leaf/root-differentiating callus culture promoted the accumulation of epoxy-pseudoisoeugenol tiglate (up to 90 mg/100 g fr.wt.) but not that of anol-derivatives. The accumulated amount of EPT in PMD-SH was comparable with that in plant seedlings.

Purified maize leaf adenylate kinase (AK) was shown to use one molecule each of free ADP and Mg-ADP as well as free AM P and Mg-ATP as substrates in the forward and reverse reaction, respectively. This was deduced from substrate kinetic studies which were carried out under conditions of strictly defined concentrations of free and Mg-complexed adenylate species and under controlled free magnesium levels. Apparent K m values of the substrates of AK were 3 and 6 μM for ADP and Mg-ADP, respectively (forward reaction), and 69 and 25 μM for free AMP and Mg-ATP, respectively (reverse reaction). The enzyme was competitively inhibited by P 1 ,P 5 -di(adenosine-5′)pentaphosphate (Ap 5 A), a bisubstrate analog of AK reaction, with apparent K i values in the range of 11 -80 nM , depending on variable substrate. Substrate kinetic studies and inhibition patterns with Ap 5 A suggested a sequential random kinetic mechanism in both directions of the reaction. These properties of a higher plant AK are similar or analogous to those previously established for the enzyme from yeast and non-plant tissues.

Conifers contain ascorbic acid which is probably a protective against oxidative attacks. In yellow needles of damaged conifers the ascorbic acid content is increased, this suggests an induction by air pollutants and/or photooxidants of a protection mechanism in trees. To study this mechanism in more detail, a green cell suspension culture of spruce (Picea abies) has been established. These cells contain chlorophyll and have a photosynthetic capacity, but they need carbohydrates (sucrose) for growth and are thus photoheterotrophic. Their ascorbic acid content and its regulation depends on carbohydrates, the cells resemble healthy green needles in this respect; they appear to be qualified for studies on the induction of a defense mechanism. Treatment of these cells with SO 2 (20 ppm, 4 days) ozone (1 ppm, 14 days) and vaporous H 2 O 2 revealed a decrease of the ascorbic acid content and (to a lesser extent) also of chlorophyll. It is concluded that (photo-)oxidants obviously do not play such a dominant role in bleaching of needles as it is assumed. The increased ascorbic acid content found in needles of damaged conifers is probably not an induced protection mechanism against oxidants.

Antibodies have been raised to synthetic peptides, corresponding to a region in the loop spanning helices 4 and 5 of D 1 protein (Ala 250-Phe 265) and to a region anticipated to be near the C terminus of mature D 1 (His 332-Ala 345). Polyclonal antibodies to the sequence His 332-Ala 345 reacted with a 32 kDa polypeptide in thylakoid preparations identified as D 1 from its resistance (pea) or susceptibility (wheat) to lysine-C degradation. A monoclonal antibody to His 332-Ala 345 reacted preferentially with a faster migrating polypeptide in SDS electrophoresis, a putative conformer of D 1. Polyclonal antibodies to the sequence Ala 250- Phe 265 also reacted with the faster running polypeptide but not with the population of molecules running at 32 kDa. The putative conformer of D 1 from wheat appears to be more resistant than the main D1 population to lysine-C degradation. Peptide analyses by Takahashi et al. [(1988) FEBS Lett, 240, 6 - 8 ] suggest Asn 335-Ala 344 lies at the processed C terminus. The present report provides immunological confirmation that this sequence is retained in mature D 1.

Treatment of PS II particles with either 1 M NaCl or alkaline Tris (1 M , pH 8.4) caused a considerable decrease in the average net negative surface charge density, concomitant with depletion of the extrinsic 17, 24 and 33 kDa proteins of the oxygen evolving complex from the membranes. The partial recovery of the values for surface charge in both NaCl- and Tris-treated membranes was registered after reconstitution experiments with the three proteins. These results are compared with the data for the charge densities of the thylakoid membranes, to examine the role of the three extrinsic proteins in the formation of heterogeneous arrangement of surface charge across the appressed (granal) thylakoids.

Affinity binding to Sepharose 4 B-immobilized ribulose-1,5-bisphosphate carboxylase (Rubisco) and its isolated small (SSU) and large (LSU) subunits of Nonidet P40-solubilized chloroplast thylakoid proteins has been performed to search for Rubisco membrane receptors in vitro. It was found that Rubisco and both of its isolated subunit types specifically bind the same proteins including H + -ATP synthase, cytochrome ƒ/b 6 complex, ferredoxin-NADP + reductase (FNR) and components of photosystem (PS) II which presumably form multienzyme complexes. PS I was not recognized by Rubisco components. of the Rubisco-binding thylakoid proteins, at least coupling factor 1 (CF 1 ) is shown to form specific complexes with the enzyme as revealed by affinity chromatography and protein hybridization on nitrocellulose filters. Employing the latter approach, the SSU and LSU of Rubisco were observed to bind independently to isolated α and β, but not to γ, δ and ε subunits of CF 1 . The results indicate that H + -ATP synthase may serve as a Rubisco membrane receptor at least in vitro and suggest that the irreversible inhibition of photophosphorylation by Rubisco [R. T. Furbank et al., Biochim. Biophys. Acta 852,46-54 (1986)] is mainly due to its binding to the α/β subunit pairs of CF 1 .

This in vitro study focuses on the UVA-induced reactions with DNA of thioridazine (TRZ), and two of its major metabolites (TRZ-2-sulfoxide or mesoridazine, MRZ ; and TRZ- 2-sulfone or sulforidazine, SRZ). TRZ binds covalently to DNA upon UVA-irradiation. Under comparable irradiation conditions, MRZ binds to a lesser extent and almost no binding was observed with SRZ. Besides, photo-induced genetic effects were investigated by means of a differential DNA repair test in E. coli. The photo-induced genetic effects in E. coli decreased from TRZ, MRZ to SRZ, which corresponds with their capacity for UVA-induced binding to DNA. TRZ, MRZ and SRZ differed in their rate of photodecomposition rather than in the intrinsic reactivity towards DNA of the instable intermediates formed. Irreversible binding to DNA was also observed upon treatment with peroxidase, which is known to oxidize phenothiazines via the formation of reactive radical cation species. As both the colour of the intermediate and its reactivity towards DNA were comparable for peroxidase treatment and exposure to UVA, we assume that the radical cation is the reactive species in the latter case as well.

The sulfhydryl enzyme malate synthase was inactivated by X-irradiation in air-saturated aqueous solution, in the absence or presence of a variety of additives (thiols, antioxienzymes, typical radical scavengers, inorganic salts, buffer components, substrates, products, analogues). Radiation-induced changes of enzymic activity were registered immediately after stop of irradiation and in the post-irradiation period. Repair experiments were initiated by postirradiation addition of dithiothreitol. Additionally, post-irradiation inactivation was modulated by some further additives. Probing the extent of primary and post-irradiation inactivation and repair was accomplished effectively by screening experiments on the microlevel, and by derivation of normalized efficiency parameters which allowed quick comparisons of the various additives with respect to their protective and repair-promotive efficiencies. Correlations between the efficiency parameters were studied by means of binary and ternary diagrams. Most of the substances added before irradiation were found to protect the enzyme against primary and post-irradiation inactivation and to increase the reparability of the enzyme by dithiothreitol, the extent of the effects depending on the nature (and concentration) of the additives used. Our results indicate that both specific protection (by substrates, products, analogues, and by sulfhydryl agents) and scavenging are responsible for the radioprotective efficiencies of the additives.

The ribonucleosides adenosine, cytidine, guanosine, inosine and uridine as well as the modified components N 1-methyladenosine and N 6 -carbamoylthreonyladenosine were characterized and determined quantitatively as minor constituents in raw bovine milk by use of an automated high performance liquid chromatography system. The studies have shown that except for the colostral phase the ribonucleoside levels are constant throughout the whole lactation period. That means, there is a typical ribonucleoside pattern which is assumed to be speciesspecific.

Previous work has shown that 1,25-dihydroxy-vitamin D 3 rapidly increases calmodulin levels of skeletal muscle membranes without altering the muscle cell calmodulin content. Therefore, the effects of the sterol on the binding of calmodulin to specific muscle membrane proteins were investigated. Soleus muscles from vitamin D-deficient chicks were treated in vitro for short intervals (5-15 min) with physiological concentrations of 1,25-dihydroxy-vitamin D 3 . Proteins of mitochondria and microsomes isolated by differential centrifugation were separated on sodium dodecyl sulfate polyacrylamide gels. Calmodulin-binding proteins were identified by a [ 125 I]calmodulin gel overlay procedure followed by autoradiography. 1,25-Dihydroxy- vitamin D 3 increased the binding of labelled calmodulin to a major, calcium-independent, calmodulin-binding protein of 28 Kda localized in microsomes, and to minor calmodulin- binding proteins of 78 and 130 Kda proteins localized in mitochondria. The binding of [ 125 I]calmodulin to these proteins was abolished by flufenazine or excess non-radioactive calmodulin. 1,25-Dihydroxy-vitamin D 3 rapidly increased muscle tissue Ca uptake and cyclic AM P levels and stimulated the phosphorylation of several membrane proteins including those whose calmodulin-binding capacity potentiates. Analogously to the sterol, forskolin increased membrane calmodulin content, calmodulin binding to the 28 Kda microsomal protein and 45 Ca uptake by soleus muscle preparations. Forskolin also induced a similar profile of changes in muscle membrane protein phosphorylation as the hormone. These results suggest that 1,25- dihydroxy-vitamin D 3 affects calmodulin distribution in muscle cells through cyclic AMP-dependent phosphorylation of membrane calmodulin-binding proteins. These changes may play a role in the stimulation of muscle Ca uptake by the sterol.

Calcium transport of skeletal muscle sarcoplasmic reticulum from golden hamsters was studied in January and in June on animals kept at 22 °C under natural photoperiod and in January after cold-acclimation at ±2 °C in the dark for 55 days. Crude homogenates from psoas and soleus muscles and from mixed skeletal muscles were used. No differences were observed in the calcium storing capacity of sarcoplasmic reticulum among the three groups of animals. Kinetic studies on the dependence of the calcium uptake rate on the concentration of free calcium revealed a significant increase of the uptake rates and a decrease of the calcium affinity in the control animals sacrificed in winter as compared to those killed in June. Cold-acclimation in winter leads to a further small reduction of the calcium affinity. This shift of calcium uptake rate and affinity in the sense of that of a fast-twitch muscle may be related to the functional demands of the cold season and cold-acclimation respectively.

The inhibitory effect of various oral doses of different hexacyanoferrate(II) compounds (HCF) and the influence of the time interval of HCF-administration on intestinal 134 Cs-absorption was studied in rats. Optimum inhibition was obtained by administration of HCF together with or 2 min before oral 134 Cs loading. Using appropriate low amounts (0.1 -0.5 mg) of the different HCF compounds, the inhibitory effect increased in the sequence KZnHCF < KCuHCF < FeHCF < KCoHCF = KNiHCF < NH 4 FeHCF = KFeHCF . Oral administration of 5 mg (0.5 mg) of KFeHCF , together with 134 CsCl loading, reduces 134 Cs-absorption from 41 % (control) to 0.8% (2.8%). Zinc-, copper-, cobalt, and nickel hexacyanoferrates(II), despite showing a high caesium sorption capacity in vitro, were less effective in rats and are not suited for in vivo application, also because they may produce toxic side effects. As a consequence, the orally administered colloidal-soluble iron (III) hexacyanoferrates(II) (NH 4 Fe[Fe(CN) 6 ] and KFe[Fe(CN) 6 ]) have to be considered as the most valuable countermeasure against radiocaesium absorption for humans and domestic animals in the case of a severe nuclear accident in the future. Manganese oxide, a non-hexacyanoferrate(II) compound with known in vitro caesium binding capacity, showed no inhibitory effect on radiocaesium absorption in rats.

“Soluble” (KFe III [Fe II (CN ) 6 ]) and “insoluble Prussian blue” (Fe III 4 [Fe II (CN) 6 ] 3 labelled with 59 Fe either in the ferric (Fe III ) or ferro (Fe II ) position and 14 C in the cyanide group were synthesized and administered intraperitoneally or orally to adult female rats with normal body iron stores. Following i.p. injection of KFe[Fe(CN) 6 ], the colloidal complex is disintegrated into ferric iron and hexacyanoferrate(II) anion almost completely. About 96% of the ferric iron was retained in the body. Nearly 90% of both ferrous iron and cyanide were excreted with the urine within 7 days after i.p. injection, indicating that most of the undissociated hexacyanoferrate(II) anion ([Fe(CN) 6 ] 4- ) was excreted through the kidney. Only 9% of the ferrous iron from [Fe(CN) 6 ] 4- was found mainly in carcass, liver and gut. As the 59 Fe/ 14 C-ratios in organs were found close to 1.0, the dissociation of the hexacyanoferrate(II) anion can only be small in vivo. No detectable 14 CO 2 -activity (< 0.01 %) was monitored in the breath of rats after i.p. injection of the 14 C-labelled KFe[Fe(CN) 6 ], also indicating that no significant amounts of cyanide were released after parenteral administration. After oral administration of the soluble and insoluble Prussian blue, 0 .3 -0 .7% of the ferric iron was absorbed and retained mainly in carcass, liver and blood. Only 0.06-0.18% of the ferrous iron was absorbed and mostly excreted with the urine (0.05-0.15%), so that only 0.01-0.03% of the oral ferrous 59 Fe was retained in the body after 7- 10 days. Very small fractions of 14 C-label from the 14 CN-group of the soluble and insoluble hexacyanoferrate(II) were observed in the exhaled air (0.04-0.08% of the oral dose). From the 14 CO 2 -exhalation, the 14 Curine excretion and the distribution of iron in blood and organs it can be concluded that the hexacyanoferrate(II) moiety disintegrated only to a small extent in the intestinal tract after oral administration. From a dose of 36 mg hexacyanoferrate(II)/kg, an amount of free (non complex bound) cyanide can be calculated which is in maximum two orders of magnitude below the LD 100 -level. Thus, the very low bioavailability of iron and cyanide from hexacyanoferrate(II) compounds after oral application is demonstrated in rats. In the case of a severe nuclear accident, appropriate doses of “soluble” and “insoluble” Prussian blue can be used as safe and effective antidote against radiocaesium contamination.

The morphology and ultrastructure of the Dufour gland of a pseudomyrmecine ant is described. The gland has an unusually large volume but possesses a very thin epithelial lining. Its morphology does not constitute a specific subfamily character as in some ant subfamilies. Analysis by gas chromatography-mass spectrometry of the glands of three species showed them all to be rich in saturated linear hydrocarbons (C 17:0 and C 15:0 dominant) and isopropyl esters of the common fatty acids. Each species produces its own characteristic mixture of these substances. The most abundant ester in Pseudomyrmex ferruginea is isopropyl oleate and in P. sp. A it is isopropyl palmitate, in P.flavicornis there is only a trace of isopropyl palmitate. An alate female of P. ferruginea contained a very similar mixture to that of the workers.

By means of closed loop Stripping the relative evaporation rates of monounsaturated C 10 -C 16 -acetates and C 10 -C 12 -alcohols have been determined. The results of stimulus released demonstrate the different volatilities of pheromone compounds which should be taken in account with certain biotests and physiological studies.

Following injection or ingestion of [ 3 H]ecdysone and [ 3 H] 2 O-hydroxyecdysone, respectively, considerable quantities of 14-deoxyecdysteroids could be extracted from gut and faeces of female adult crickets. The compounds were identified as 14-deoxyecdysone and 14-deoxy-20-hydroxyecdysone on the basis of comigration with reference substances in two HPLC systems and by its mass spectrum (only 14-deoxy-20-hydroxyecdysone). The 14-dehydroxylation is most probably caused by bacteria from the gut lumen. Feeding of [ 3 H]ecdysone resulted in the appearance of labelled 14-deoxyecdysone also in ovaries, fat body, muscles, and rest of the body.

In the primitively eusocial sweat bee, Lasioglossum malachurum, 66 volatile compounds could be identified from queen Dufour’s gland secretions. The patterns found in gynes and in old nesting queens differed in the absolute amounts of extractable volatiles as well as in the relative proportions. 3-Methyl-3-butenyl octadecanoate is the main component in gynes, while 18-octadecanolide, 20-eicosanolide and 22-docosanolide largely dominate the bouquets of old queens. The probable roles of some specific compounds in pheromonal communication of mates and in nest recognition are discussed. Correlations with the volume of the fat body and the vitellogenic status of the ovary are described.

The dorsal epithelium of the plate-like primitive metazoon Trichoplax adhaerens is a sheet of flat cells, each bearing a single flagellum. This layer rests largely on an intercellular fluid and is predominantly fixed at the edges of the organism but also locally by fibre cells constituting the middle layer of the animal. Within these extensile dorsal cells the counterforce of the flagellar power stroke acting on the basal bodies subjects nearby objects to oscillatory motions. Given certain mechanical conditions, the vibrating single basal body can be compared with a springy oarlock that is rhythmically displaced with the power stroke and recoils during the recovery stroke. The basal body transmits its motion to the adjacent viscoelastic cytoplasm in which naturally coloured inclusion bodies serve as markers of the pulsating deformation visible in the living animal. These inclusions vibrate in the frequency range of the flagellar beat and stop as soon as the flagella are paralyzed by chemical means. That the vibrations can only intermittently be observed may be due to the varying tension of the dorsal epithelium exposed to the forces of the contractile fibre cells. -The rhythmic cytoplasmic deformation synchronous with the flagellar action has apparently not yet been described in metazoa and could be documented by video recording.

A recent model of landmark orientation by the bee assumes that the memory of the landmarks is arranged in a kind of a mental map. Our experiments disprove this assumption and show that the sun compass dominates the orientation without any indication of mental operations within a map-like representation of landmarks or of compass vectors and distances.

Peganum harmala, Hairy Root Cultures, β-Carboline Alkaloids Hairy root cultures of Peganum harmala were established by genetic transformation with Agrobacterium rhizogenes strain A4. In contrast to suspension cultures the root cultures contained high levels of β-carboline alka­ loids (1 -1.5% of dry mass).

Micellar aggregates have been prepared from chloro­phyll a. Whereas most detergents including non-ionic, zwitterionic and anionic ones, produce aggregates with long-wavelength shifted and enhanced Q Y absorption, the cationic detergents hexadecyl-trimethylammonium-bromide and hexadecyl-pyridiniumchloride produce complexes with opposite traits: The Q Y absorption is shifted to shorter wavelengths, and it is hypochromic. The optical activity is increased as in the long-wave­ length shifted complexes, but the cd bands are non-conservative and coincide with the absorption maxima. The complexes are most likely small aggregates of chlorophyll with a changed geometry as compared to the “standard” ones.