Band 43, Heft 9-10

The rates of aryl sulfatase hydrolysis of several 7-, 4′- and 3-sulfated flavonoids were compared and found to follow the order 7 or 4′ >>> 3. The complete resistance of the 3-sulfate ester to enzyme hydrolysis provided a unique and convenient method for the synthesis of a number of naturally occurring flavonol-3-sulfates from the corresponding higher sulfated analogs in quantitative yield.

A number of flavonoid 3′,4′-disulfates were synthesized from the corresponding 4′-sulfate esters, using sulfur trioxide-trimethylamine complex. Desulfation of the sulfate esters using aryl sulfatase demonstrated that the rate of hydrolysis of the 3′-sulfate group was slower than either the 7- or 4′ groups, thus allowing the specific synthesis of flavonol 3,3′-disulfates. The effects of ortho-disulfation on the 13 C NMR spectra of flavonoids, and the regative FAB-MS spectra of diand trisulfated flavonoids are discussed.

Callus cultures of various cultivars of Vicia faba were studied for regeneration. During callus growth on agar or in liquid medium a black tissue was often observed correlated with an autolysis of some cells. With those tissues callus growth and regeneration of plants was inhibited. In black callus the phytoalexin synthesis is induced without infection by microorganism. The main phytoalexins in the callus tissue are wyerol and wyerone, with some wyeronic acid and dihydrowyerone. Wyerol was found especially in the cultivar Troy at a high concentration. In four cultivars the pooled phytoalexin concentration varied between 10 and 150 μg · g -1 fresh weight.

The authors of Tulipa cv. Apeldoorn accumulate a large amount of ferulic acid sucrose esters. In addition to the well-known diferuloyl-(DFS) and triferuloyl sucrose esters (TFS), a new phenylpropanoid characterized as a monoester of ferulic acid and sucrose is described. Like T F S and D F S , this ester exhibits an accumulation m a x i m u m in the early stages of pollen development. Numerous free sugars have been extracted from Tulipa anthers. Three of them were identified as sucrose, glucose and fructose. Sucrose as the main component is accumulated in extremely high amounts during specific developmental stages (= 20 % of the dry weight). After separation of the anthers into an anther wall fraction (AWF) and pollentapetum (PTF), each fraction shows a different accumulation kinetic of the free sugars and the ferulic acid esters. A correlation could be observed between the occurrence of the esters and sucrose in the pollentapetum fraction during the anther development.

Protein extracts from anthers of Tulipa cv. Apeldoorn catalyze the degradation of ferulic acid sucrose esters. Different products are generated when triferuloyl sucrose (TFS) and diferuloyl sucrose (DFS) were applied as substrates. By the aid of reversed-phase HPLC , TLC and spectroscopy the products could be identified as free ferulic acid, monoferuloyl sucrose ester [feruloylsucrose(mono)] and two different diesters of ferulic acid and sucrose [feruloylsucrose(di) and the endogenously occurring diferuloylsucrose (DFS)]. By means of protein fractionation (chromatofocusing, anion exchange HPLC and molecular sieving HPLC) , four different enzyme activities involved in the degradation process could be separated. According to their catalytic properties, they were characterized as esterases (= EA). The partially purified esterase activity I (EAI) obtained after fractionation by chromatofocusing catalyzes the formation of feruloylsucrose(di) and ferulic acid when TFS is used as substrate. Incubations with EA la or EA Ib isolated in smaller portions lead to the same product pattern. The esterase activity II (EA II) degrades TFS to ferulic acid and DFS . DFS as substrate is only accepted by the EA I activities, in all three cases ferulic acid and feruloyl sucrose(mono) are formed as products. The kinds of different degradation reactions clearly indicate that one enzyme (= the EA II activity) catalyzes exclusively the formation of DFS from TFS. Both enzymes, EA I and EA II, exhibit a high specificity towards ferulic acid sucrose esters. Hydroxycinnamic sucrose esters with only sinapic acid moieties do not function as substrates. When enzymatically formed sucrose esters like feruloylsucrose(di), feruloylsucrose(mono) and monosinapoylsucrose were used as substrates, no product formation could be observed. Applying SFS as substrate, only the ferulic acid moiety was released by EA I. Further, naturally occurring esters (glucose- and CoA-esters of p-coumaric, caffeic, ferulic and sinapic acid; chlorogenic acid; BGM ) tested so far were not degraded by EA I and EA II. It is assumed that these esterase activities play a specific role in the ferulic acid metabolism in Tulipa anthers.

The binding capacity of penicillin G-sulfoxide towards the penicillin-binding proteins (PBP) of Staphylococcus aureus H was studied. The sulfoxide and its parent compound, penicillin G , differ only in two aspects, the sulfur-bound oxygen and an altered conformation of the five-membered thiazolidine-ring system. These minor alterations of the penicillin structure resulted in a drastical decrease of binding activity (about two orders of magnitude) of the sulfoxide derivative towards its target enzymes. Furthermore, the sulfoxide did not exhibit the selectivity of subinhibitory doses for PBP 3, as could be observed for penicillin G . The biological consequences of this behaviour were monitored via growth curves, uptake of cell wall label, and analysis of the cell wall. Binding studies revealed that comparable growth inhibition and impairment of cell wall label uptake were achieved by at least a 100-fold higher penicillin G-sulfoxide concentration, compared to its parent compound. In cell wall analysis, the application of high doses of the antibiotics, i.e. nearly saturated PBP , verified the above mentioned observation. Surprisingly, small but significant differences in cell wall composition occurred using subinhibitory doses, probably due to the altered affinity towards PBP 3, supporting the hypothesis of an important role of this PBP in peptidoglycan transpeptidation.

1-O-hexadecyl-2-acetyl-sn-glyceryl-3-phosphorylcholine (platelet activating factor, PAF), (1.9 μmol) was prepared from the total lipid extract of the protozoan Tetrahymena pyriformis 9 × 10 7 cells. The procedure involved mild alkaline hydrolysis of the total lipids, followed by acetylation and purification of the product by preparative TLC and HPLC . The yield was 60 % with respect to the content of 1-O-alkyl-2-acyl-sn-glyceryl-3-phosphorylcholine in the total lipids, determined after preparative TLC . The alkyl side chain of the semisynthetic PAF was composed of hexadecyl residue. Our product was identified as PAF according to its biological activity, the chromatographic behaviour on TLC and HPLC , the physicochemical properties and the behaviour under treatment with PLA 2 and Lipase from Rhizopus arrhizus. The above procedure is proposed as a facile, inexpensive and convenient method.

The state of the tryptophan residues of porcine kidney aminoacylase I (EC 3.5.1.14) was investigated by fluorescence spectroscopy and chemical modification. The pH-dependence of the fluorescence emission spectrum of the enzyme indicates that its native conformation prevails between pH 6 and 9.5. Within this range, the ionization of a residue with an apparent pKa of 7.1 quenches the enzyme fluorescence by about 15%. A similar reduction of fluorescence intensity accompanies the inactivation of aminoacylase I by treatment with N-bromosuccinimide in low excess. This suggests that in both cases a single tryptophyl residue out of eight residues per subunit is affected. Quenching by iodide revealed that, in the native conformation of the enzyme, 5-6 tryptophans per subunit are accessible, while 2-3 are buried within the protein. 8-Anilinonaphthalene- L-sulfonate (ANS) is tightly bound to aminoacylase I (1 mol/mol dimer, K d < 1 μᴍ). ANS binding does not interfere with substrate turnover; the spectroscopic properties of the aminoacylase- ANS complex are consistent with bound ANS being excited by radiationless energy transfer (RET) from buried tryptophyl residues of the enzyme.

A calcium-activated neutral proteinase (CANP) was prepared from the soluble fraction of calf thymus and purified to electrophoretical homogeneity. The purified proteinase was shown to consist of two subunits, each of 80 kDa, in contrast to rabbit skeletal muscle calpain which was shown to consist of 80 kDa and 30 kDa subunits. The calcium requirement for 50 % activation was 0.55 mᴍ, indicating that this enzyme belongs to the low calcium sensitive type of CANP , named mCANP or Calpain II. Optimal conditions of enzyme activity towards 0.8% casein as substrate are pH 7.5, a calcium concentration of 1.5 mᴍ, the presence of an SH-reducing agent and an incubation temperature of 30 °C. The enzyme is inhibited by Zn 2+ , p-chloromercuribenzoate and N-ethylmaleimide.

A new measuring system for monitoring absorbance changes around 830 n m is described, which was developed by modification of a commercially available pulse modulation fluorometer. All modifications concern the emitter-detector unit of the fluorometer, such that only this unit needs to be exchanged when changing from fluorescence to absorbance measurements and vice versa. The new system is shown to be well-suited for measuring redox changes of P700, the reaction center of photosystem I, in intact leaves and isolated chloroplasts. The observed kinetic changes at 830 nm in response to single turnover or multiple turnover saturating flashes are practically identical to those previously measured around 700 nm . The signal/noise ratio is sufficiently high to give well-resolved kinetics without signal averaging. W h e n P700 is oxidized by far-red background light, valuable information on the state of the intersystem electron transport chain is given by the re-reduction kinetics induced by single or multiple turnover saturating flashes. Such measurements are facilitated by the use of poly-furcated fiberoptics. With intact leaves, almost identical responses are found when measuring through the leaf (transmission mode) or from the leaf surface (remission mode). Modulated chlorophyll fluorescence can be measured in parallel; application of saturation pulses for fluorescence quenching analysis produces transient P700 oxidation without oversaturating the measuring system. The information on the P700 redox state complements that obtained from fluorescence measurements, yielding a new practical tool in plant physiological research.

Peroxidizing herbicides were assayed for their capacity to accumulate tetrapyrrole-like compounds in the liquid culture medium when growing the xanthophycean alga Bumilleriopsis filiformis. We have isolated and purified this compound by HPLC . For identification optical, fluorescence, NMR and mass spectroscopy were employed. All these techniques showed that the accumulation product is protoporphyrin IX. This tetrapyrrole was found, although to a different extent, using either chlorophthalim, oxadiazon, oxyfluorfen or LS 82-556. Accumulation of protoporphyrin IX caused by peroxidizing compounds was also found in light-grown Bumilleriopsis cells and heterotrophically (dark-)grown Scenedesmus cells, but neither in light-grown Scenedesmus cells nor in dark-grown Neurospora.

A number of 25 nitro-diphenylethers have been synthesized and assayed for their inhibitory activity in N-methylphenazonium-methosulfate mediated cyclic photophosphorylation. Their pI 50 -values were in the range from 3.5 to 5.3. A QSAR-analysis revealed that the lipophilicity of the nitro-diphenylethers as expressed by ∑π played a major role. In addition, the Taft steric parameter E s in positions 6′, 4′ and 2′ (in decreasing order) contributed to the inhibitory activity. For maximal biological activity, positions 4′ and 6′ should be left unsubstituted whereas a bulky substituent is required for position 2′.

Oxygen gas exchange was monitored in the unicellular green alga Chlorella vulgaris 211 - 11 h by means of a mass spectrometer equipped with a special membrane gas-inlet-system and a photosynthetic reaction vessel. CO 2 -dependent 18 O 2 -uptake as well as 16 O 2 -evolution were analyzed in both High- and Low-CO 2 cells. In High-CO 2 cells, the 18 O 2 -ruptake in the light (U L ) decreased by 65% upon addition of 3 mᴍ NaHCO 3 , while 16 O 2 -evolution (E) was increased approx. 1.8 times by the same treatment. 18 O 2 -uptake in the dark (U D ) was not affected by the addition of external inorganic carbon (Ci). The addition of 3.3 mᴍ NaHCO 3 also affected U L and E in Low CO 2 -cells, however, to a minor extent. U L under CO 2 -saturating conditions was light intensity-independent up to 2 klux and 1.2 klux in High- and Low-CO 2 cells, respectively. Above these light intensities U L increased approx. 4-fold in High- and approx. 6-fold in Low-CO 2 cells. Under CO 2 -limiting conditions, however, U L increased in High-CO 2 cells even under very low light intensities, showing that photorespiratory oxygen uptake occurred even in the near vicinity of the light compensation point. Under C02-saturating and strong light conditions U L represented almost half of E in Low-CO 2 cells and about 30 % of E in High-CO 2 cells. In Low-CO 2 cells addition of ethoxyzolamide (EZA), an inhibitor of carbonic anhydrase, enhanced U L and suppressed E and NET under CO 2 -limiting conditions, whereas the compound had only a minor effect on High-CO 2 cells. DCMU (3 μᴍ) strongly inhibited E and U L under CO 2 -saturating conditions, with the remaining U L being smaller than U D . KCN (1 mᴍ) and SHAM (1.5 mᴍ) added to DCMU-treated Low-CO 2 cells suppressed U L by approx. 50 % . The resulting value corresponded to half of U D . KCN also inhibited E under CO 2 -saturating conditions, with U L being strongly enhanced showing a maximal uptake at 0.4 mᴍ KCN . Under these conditions NET was nearly zero. The effect seems to be due to an inhibition of RubisCO and an enhancement of Mehler reactions. At 0.7 mᴍ KCN , DCMU entirely inhibited U L , but oxygen uptake appeared increased after turning the light off. This uptake corresponded to approx. 60 % of U D . Whereas KCN and SHAM inhibited approx. 70 % of U D , only 16% of U L was suppressed. These results suggest that the contribution of mitochondrial respiration to U L was negligeable, since U L seemed to be suppressed in the light under CO 2 -saturated conditions. Iodoacetamide, which is an inhibitor of the Calvin cycle and thereby diverts carbon into the respiratory pathway, inhibited E and NET under CO 2 -saturating conditions, but did not affect U L . This result also shows that U L is not due to mitochondrial respiration. A hydroxylamine derivative [20, 21] which changes the ratio of the RuBP carboxylation to oxygenation activity in tobacco leaves did not affect this ratio in Chlorella.

In the present paper we have attempted to characterize by means of immunological methods the molecular difference between ribulose 1,5-bisphosphate carboxylase/oxygenase from the wild type TV. tabacum var. John William's Broadleaf and the tobacco mutant Su/su. The tobacco mutant Su/su exhibits in comparison to the wild type a higher photorespiratory activity. The reagents used in the present study are monospecific antisera to the two bifunctional enzymes to be compared. We have analyzed the oxygenase activity of the two enzymes in dependance on the binding of the amount of antibodies out of the homologous and the not homologous antiserum. These analyses have shown, that the enzymes of both phenotypes were 4 0 % stronger inhibited in the equivalence regions with respect to their oxygenase function by antibodies of the antiserum to ribulose 1,5-bisphosphate carboxylase/oxygenase of the mutant than by antibodies of the wild type antiserum. It should be noted that the antiserum to the mutant enzyme exhibits a 25 % lower antibody titer, than the antiserum to the wild type enzyme. In the region of extreme antibody excess, i.e. when antibodies are mostly monovalently bound and most antigenic determinants are saturated with antibodies, the oxygenase activity of both enzymes decreases towards zero in the presence of the homologous as well as in the presence of non-homologous antibodies. In the region of excess of antigenic determinants, that is when only a few antibody molecules can bind onto the enzymes, only the oxygenase activity of the mutant enzyme is inhibited by its homologous antibodies by 40 % . This apparent difference in the molecular structure of the two bifunctional enzymes to be compared is neither caught in the double immuno diffussion test nor in the tandem crossed immuno electrophoresis, using the two antisera as test reagents. In all cases only fused precipitation bands are observed. Chemical modification of ribulose 1,5-bisphosphate carboxylase/oxygenase by hydroxylamine treatment or treatment with o-(p-nitrophenyl)hydroxylamine or simple heating of the enzyme to 50 °C are immunologically characterized. As a consequence of such treatment considerably less antibodies are adsorbed. The strongest influence exerts a treatment with o-(p-nitrophenyl)-hydroxylamine with simultaneous heat treatment of the enzyme for 20 min at 50 °C.

Specific and stoichiometric binding is shown between the D-1/D-2 sub-core complex and the 33 kDa polypeptide of photosystem II. Fluorescence from chlorophyll is used as an endogenous probe. W h e n binding occurs there is an increase in fluorescence yield, as well as changes in both the fluorescence spectrum and excitation spectrum.

2-Isobutyl-1 ,3-thiazole and its 4,5-dihydro derivative were identified in the preorbital gland secretions of the grey duiker, Sylvicapra grimmia, and the red duiker, Cephalophus natalensis, but are absent from the preorbital secretion of the blue duiker, C. monticola. These two compounds which are present in high, but varying concentrations in the secretions of male grey duikers, are present in low concentrations in the secretions of females. This seems to be the only consistent significant difference between the secretions of male and female grey duikers and together with the fact that only males mark out their territories, was construed as evidence in favour of these two compounds playing a significant role in the territorial behaviour of male grey duikers.

In larvae and later developmental stages of Creatonotos transiens, which had been reared on the pyrrolizidine alkaloid 7S-heliotrine, a new major metabolite was detected by capillary GLC. The structure of this metabolite was determined by GLC-MS (EI, CI-MS) and 13 C NMR to be 7Rheliotrine and 7R-heliotrine-N-oxide. 7R-Heliotrine is likely to be the direct precursor for the pheromone R( - )-hydroxydanaidal.

Chlorpromazine is a widely used phenothiazine tranquilizer known to alter the shape of normal erythrocytes and their osmotic fragility. In order to understand the nature of the interactions chlorpromazine · HCl (CPZ · HCl) was made to interact with phospholipid bilayers formed by dimyristoylphosphatidylethanolamine (DMPE) and dimyristoylphosphatidylcholine (DMPC). This study was carried out by X-ray diffraction on crystalline powders of various molar mixtures of CPZ · HCl with DMPE and DMPC, with and without water. It was found that CPZ · HCl significatively affects the bilayer structure of DMPC in the presence of water, but not that of DMPE.

Several methods for the preparation of “white ghosts” or “resealed ghosts” were described in the recent literature. This article compares three methods to prepare white, resealed ghosts from bovine erythrocytes based on the principle of hypotonic lysis. The methods described differ by the removal of hemoglobin from the empty cells. The main difference between the standard centrifugation, the gelfiltration and the hollow-fibre diafiltration is the mechanical stress on the leaky membranes after swelling in hypotonic media. Mean cellular volumes, rates of potassium-efflux and the access of impermeable dyes to cytoplasmatic proteins are criteria to differentiate between ghostcell-populations.

The movement of micro-organisms was investigated in different cellular environments. The type of movement was described in terms of the symmetry of the cellular environment: (i) Random movement - isotropic symmetry of the environment, (ii) contact guidance - apolar symmetry of the environment and (iii) directed movement as Chemotaxis and galvanotaxis - polar symmetry of the environment. To quantitate cell movement it was necessary to parameterize the environment as well as the cell movement by observables. The random movement was quantitated by the diffusion coefficient. The contact guidance of the nematic type and the contact guidance on a bent surface were quantified by an apolar order parameter. The contact guidance constant for fibroblast on a glass cylinder was 87.5 pm. The directed movement was quantified by a polar order parameter. Dose-response curves were derived and compared for different types of cells: Chemotaxis of granulocytes K CT -1 = 2.2 mm for 10 μᴍ f-Met-Met-Met, galvanotaxis of granulocytes K G -1 = 0.2 V/mm, galvanotaxis of fibroblast K G -1 = 0.28 V/mm, galvanotaxis of spermatozoids of bracken fern K G -1 =0.024 V/mm.

The method involves impregnation of fixed embryos with a solution of plexiglass. The solvent is evaporated slowly. Blocks of plexiglass containing the specimen are thus formed. Sections (1-2 μm) are stretched and attached by an unconvential method. Immunohistological techniques allow antigen localization in a combination of high resolution, excellent quality of the sections and low background.

The adhesion of granulocytes to a glass surface is both theoretically and experimentally investigated. The basic results are: (i) The adherence process of cells to a surface can be described by the Langmuir adsorption isotherm which also holds for the adsorption of molecules to a substrate. (ii) The granulocytes do not interact with each other during the adsorption/desorption process. (iii) The equilibrium constant at 37 °C for granulocytes adsorption to the glass surface is 170 cells/mm 3 = 1.7 × 10 5 cells/ml. (iv) The equilibrium constant increases with decreasing temperature, (v) The equilibrium constant is a function of the chemokinetic/chemotactic stimulus f-Met-Leu-Phe. (vi) The desorption rate is also a function of the chemokinetic/chemotactic stimulus (k de -1 = 71.5 min for 1 nᴍ and 330 min for 1 μᴍ f-Met-Leu-Phe). (vii) The adsorption rate is a function of the chemokinetic/chemotactic stimulus and the cellular bulk concentration (k ad -1 - · c v -1 = 104 min for 1 nᴍ and 8 min for 1 μᴍ f-Met-Leu-Phe, and c v = 4000 cells/mm 3 = 4 × 10 6 cells/ml), (viii) The total number of cells which can bind to the surface is independent of temperature (and of the chemokinetic/chemotactic stimulus), (ix) The high affinity receptor site is responsible for the adherence (K f-Met- = 1.8 nᴍ f-Met-Leu-Phe). (x) The chemokinetic/chemotactic stimulated adherence is a cooperative process on a molecular level, (xi) The cellular adsorption/desorption process is a rate controlled process. The thermodynamic description of the adsorption/desorption process failed.

Chaos dynamics, which characterizes biological information processing, generates information along the course of temporal development of the relevant system. In this system, the macroscopic uncertainty principle holds between observation time Δt and phase space volume ΔΩ determined by this observation. In other words Δt and ΔΩ cannot simultaneously be small. This principle corresponds to the microscopic uncertainty principle that holds in quantum physics. Through an analogy to this correspondence, it is shown that quantum logic might also govern such macroscopic phenomena as are governed by chaos dynamics.

Malnutrition is a common finding in Pakistan especially in children of low socio-economic class. Impairment of digestion and absorption makes the diet inefficient at fulfilling the requirements of the child and leads to malnutrition. Earlier work from this laboratory has shown that feeding a high-protein diet to children suffering from protein-energy malnutrition (PEM) did not improve their condition. In the present study forty-two malnourished children and seventeen control subjects were investigated. They were divided into five groups, i.e. fibrocystic disease of the pancreas (n = 9), coeliac disease (n = 17), lactose intolerance (n = 5), PEM (n = 5) and nonspecific diarrhoea (n = 6) on the basis of history, clinical impression and biochemical findings. They were from 6 months to 12 years of age. The majority of them were suffering from diarrhoea, distension of the abdomen and retardation of growth. All children were physically examined and anthropometric measurements were recorded. Changes in hair in the form of sparseness, dyspigmentation and easy pluckability were observed in all groups. In PEM, 80% of the cases had enlarged livers. All children had reduced weight and height as compared with the control group. Haemoglobin, total protein and serum albumin showed a decrease and the fall was greatest in PEM cases as compared with the control group. The excretion of a-amino nitrogen increased in PEM children while hydroxyproline and creatinine levels showed a decrease as compared with the control group. The hydroxyproline index, which signifies the growth rate, was also significantly lower in all groups. Xylose and lactose tolerance tests were performed after an oral dose of xylose or lactose respectively. The xylose concentration after the test dose was reduced with the lowest values in the coeliac disease group as compared with the control group. In the lactose tolerance test, the patients with lactose intolerance showed lower values as compared with control subjects. Sodium and chloride in sweat showed a significant rise in fibrocystic disease while daily faecal fat excretion was increased in fibrocystic disease, coeliac disease and lactose intolerance cases as compared with the control group. The various biochemical indicies studied indicate that malabsorption played an important role in malnourishment.

5,9-Dimethylheptadecane and 5,9-dimethylpentadecane represent major components of the sex pheromones of females of the leafminer moths Leucoptera scitella and Perileucoptera coffeella respectively; 5,9-dimethylhexadecane was found in both species as a minor component. These branched chiral hydrocarbons represent a new type of Lepidopteran pheromones, for which simple syntheses are described.

(E)-11-Hexadecenyl acetate is the major volatile component of the sex pheromone gland of female Leucinodis orbonalis collected from Sri Lanka. (E)-11-hexadecen-1-ol was also found in trace quantities.

Data are presented showing the result of the functioning of the mitochondrial respiratory chain in Ehrlich ascites tumour cells incubated with glucose and glutamine. The very different results obtained depending on the order of substrate addition suggest that the functioning of the respiratory chain in Ehrlich cells far-from-equilibrium maintains an intracellular dissipative structure.