Comparative freezing patterns of bark and xylem of “Siberian C” and “Redhaven” peach twigs

An electrophoretic mobility technique was used to study freezing patterns in excised peach twigs. Moisture content was the only qualitative difference in initial freezing patterns of similar tissues of Redhaven and Siberian C. Siberian C contained up to 18% less moisture than Redhaven. Major differences in the shape of the transition pattern were detected between bark and xylem. Even though bark tissues had twice the water content of xylem, the bark exhibited equilibrium freezing while the xylem underwent nonequilibrium freezing. Bark water must be intimately associated with the living protoplast, while xylem water is less closely associated with cellular components. Comparison of bark and xylem freezing curves with sucrose and cellulose model systems suggested that bark freezing was similar to the sucrose model while xylem freezing was similar to the cellulose model.     

The localization and the isoenzymes of α- and β-Galactosidases in root tips

The localization was studied of α- and β-galactosidases in frozen sections of Ca-formol fixed root tips using simultaneous azocoupling reaction. In all species studied (Allium cepa,Cucurbita maxima, Lupinus albus, Pisum sativum, Vicia faba, Zea mays) positive results were obtained, the localization being ubiquitous (according to localization typology given here). InVicia faba andZea mays the isoenzymes of α- and β-galactosidases were revealed by means of acrylamide gel electrophoresis, using authors’ modification of Reisfeld method, in whole root tips, particular growth zones and separately in cortex and central cylinder. No differences were observed comparing stele and cortex. Whereas characteristic isoenzyme patterns were found in individual growth zones in maize, no differences appeared in broad bean. A comparison was made of thein situ localization and of the isoenzyme patterns of α- and β-galactosidases with α- and β-glucosidases. In the case of galactosidases, positive results appear with both α- and β-galactoside. The rising of pH to neutrality leads to considerable decrease in the activity of both galactosidases.     

Selective cytochemical localization of peroxidase,cytochrome oxidase and catalase in rat liver with 3,3′-diaminobenzidine

Summary In rat liver, three different enzymes with peroxidatic activity are demonstrated with modifications of the DAB-technique: peroxidase in the endoplasmic reticulum of Kupffer cells, catalase in peroxisomes and cytochrome oxidase in mitochondria. The major problem of the DAB-methods is their limited specifity so that often in tissues incubated for one enzyme the other two proteins are also stained simultaneously. We have studied the conditions for selective staining of each of these three enzymes in rat liver fixed either by perfusion with glutaraldehyde or by immersion in a modified Karnovsky's glutaraldehyde-formaldehyde fixative. The observations indicate that in perfusion fixed material selective staining can be obtained by reduction of the incubation time (5 min) and the use of optimal conditions for each enzyme. In livers fixed by immersion the distribution of the staining is patchy and irregular and usually longer incubation times (15–30 min) are required. Selective staining of peroxidase in Kupffer cells was obtained by brief incubation at room temperature in a medium containing 2.5 mM DAB in cacodylate buffer pH 6.5 and 0.02% H₂O₂. The exclusive staining for cytochrome oxidase in cristae of mitochondria was achieved after short incubation in 2.5 mM DAB in phosphate buffer pH 7.2 containing 0.05% cytochrome c. For selective demonstration of catalase in peroxisomes the tissue was incubated in 5 mM DAB in Teorell-Stenhagen (or glycine-NaOH) butffer at pH 10.5 and 0.15% H₂O₂. The prolongation of the incubation time in peroxidase medium caused marked staining of both mitochondria and peroxisomes. In the cytochrome oxidase medium longer incubations led to slight staining of peroxisomes. The catalase medium was quite selective for this enzyme so that even after incubation for 120 min only peroxisomes stained.     

Induction of isoenzymes of alcohol dehydrogenase in “flor” yeast

Summary Two isoenzymes of alcohol dehydrogenase (adh I and adh II) from Saccharomyces cheresiensis have been differentiated by thermal treatment of the crude extracts. The effect of pH on the stability and the K _m for ethanol are different for the two isoenzymes.The proportions in which they are present depend on the carbon source used by the yeast: adh I is the major component in cells grown on glucose, and adh II in those grown on ethanol. Cells grown on glucose plus ethanol show high levels of both isoenzymes, indicating that the synthesis of adh I is subjected to nutritional induction by glucose, and that of adh II by ethanol.The physiological roles of the two isoenzymes are discussed in relation with the nutritional characteristics of S. cheresiensis.     

Developmental dissociation of myelin synthesis and “myelin-associated” enzyme activities in the shiverer mouse

Developmental changes in three enzymes associated with myelin lipids were studied in the shiverer mouse, a murine mutant showing a severe deficiency of CNS myelin. Age-related changes in cerebroside sulfotransferase (measured in brain) and arylsulfatase A and cerebroside B-galactosidase (measured in brain and liver) were the same for shiverer and control mice. The shiverer mouse, therefore, demonstrates a dissociation between the genetic mechanisms regulating myelination in the CNS and developmental changes in enzyme activities thought to be closely related to the synthesis of myelin. In addition, we found no defect in the shiverer mouse in the incorporation of glycine-labeled basic protein into CNS myelin, indicating an important metabolic difference between the morphologically similar shiverer and quaking mutants.     

Investigating the role of potassium and urea to control fruit drop and to improve fruit quality of “Dhakki” date palm

Fruit drop is a key issue with date palm cultivars that can be addressed with a variety of methods and strategies. Foliar application of macronutrients can be more effective in inhibiting fruit drop and improving the quality of date fruits. The current study was carried out to investigate the possible role of potassium (K) and urea to reduce fruit drop and improve the fruit quality of “Dhakki” date palm. It was conducted in a complete randomised block design with seven treatments and three replications at Pakistan's Agricultural Research Institute, Dera Ismail Khan. The treatments used were: (i) Control (distilled water spray); (ii) Potassium sulphate (K₂SO₄) at 1 %; (iii) K₂SO₄ at 1 % + Urea at 2 %; (iv) K₂SO₄ at 2 %; (v) K₂SO₄ at 2 % + Urea at 2 %; (vi) K₂SO₄ at 3 % and; (vii) K₂SO₄ at 3 % + Urea at 2 %. All the concentrations were sprayed at Kimri stage of fruit development during two consecutive growing seasons. Twenty-one date palms of equal size and age were chosen for the assessments to measure percent fruit drop and other physicochemical variables, including fruit length, fruit diameter, fruit weight, pulp percentage, yield/bundle, pH, total soluble solids (TSS), K content in fruit, and all sugars (percent) of harvested date fruit. The results revealed that bunch spray of K significantly affected all the parameters during both seasons. Application of K₂SO₄ alone and in combination with urea not only effectively reduced the fruit drop but also improved fruit quality in date where, K₂SO₄ applied at 2 % combined with urea was the best concentration in reducing fruit drop, enhancing other physicochemical attributes, and improving fruit quality of “Dhakki” date palm. This study may effectively contribute to reduce the fruit drop and enhance the fruit quality by using K and urea, enabling farmers to improve the date yield and increase economic growth.     

Comparative studies on the activities of chitinase, β-1,3-glucanase, peroxidase and phenylalanine ammonia lyase in the leaves of triticale and wheat infected with Stagonospora nodorum

The activities of chitinase, β-1,3-glucanase, peroxidase and phenylalanine ammonia lyase, constitutive and induced by Stagonospora nodorum were examined in the 10 – 14 day old seedlings of three triticale and two wheat cultivars under controlled environmental conditions and in flag leaves of two triticale cultivars in the field. Two S. nodorum isolates of different virulence were used. Both the constitutive and induced activities in triticale and wheat depended on genotype and in triticale the effect of growth conditions was also evidenced. The constitutive activities of chitinase, β-1,3-glucanase and peroxidase were several fold lower in flag triticale leaves in plants from the field than in the seedlings, growing under controlled conditions, but induction in the infected flag leaves was significantly more pronounced. In triticale genotypic differences in the response to infection were revealed only upon inoculation by S. nodorum isolate of higher virulence. The enzymatic activities increased several fold during successive days after the infection except for phenylalanine ammonia lyase. Induction of this enzyme was only transient and the activity decreased 48 or 96 h after infection when the activities of other enzymes were rising. In flag leaves in the field this activity was differentiated only after infection with more a virulent strain. A tendency appeared in triticale seedlings for association of the resistance to the pathogen with lower enzymatic constitutive activities. This relationship became more evident in triticale infected by S. nodorum and may imply that although the investigated enzymes are certainly involved in general, non-specific defense mechanism, they do not decide on the resistance to pathogen at least in the early stages of infection and cooperate with other factors in the complex pathogen-plant interaction. One can also assume that the enzymatic activities are associated with severity of infection rather than resistance to pathogen.     

The relation between the levels of extractable and diffusible IAA in almond fruits and their “June drop”

The relationship between the June drop of almond fruits(cv. Truoito) and the levels of extractable and diffusible IAA of the fruit wasstudied. June drop started almost simultaneously with thedecreasein the concentration of extractable free and ester IAA from seeds and theamountof diffusible free IAA from persisting fruits. The drop lasted for a period ofabout 17 days, during which time the auxin decreased constantly. In a secondexperiment, the relationship between fruit size, fruit drop and level of auxinwas examined. The fruits were classified according to size into threecategories, large, intermediate and small. Throughout the experiment thepercentage of small fruits that abscised (70%) was five times higher than thatof the other categories of fruits. The small persisting fruits had lower levelsof extractable free and diffusible free IAA than fruits of intermediate andlarge size. When the process of abscission advanced and the diffusion of theauxin had been interrupted, an increase in the concentration of extractablefreeand peptidic IAA in the seeds and pericarps of the abscising fruits wasobservedcompared with the persisting fruits.     

Accumulation of the β-subunit of polygalacturonase 1 in normal and mutant tomato fruit

Polyclonal antiserum raised against the native PG1 isoform of tomato fruit (Lycopersicon esculentum Mill.) polygalacturonase [poly(1,4--d-galacturonide) glycanohydrolase, EC 3.2.1.15] bound to each of the subunits of the protein and also to a range of other fruit proteins. Affinity purification was used to remove antibody molecules that bound to the native form of the PG2 isoform. The resulting serum bound to native PG1, denatured PG2 and -subunits of PG1 but not to native PG2 or other fruit proteins. This anti-PG1 serum was used to monitor the occurrence of the PG1 -subunit and PG2 in detergent extracts of tomato tissues. The -subunit polypeptide was detected in pericarp but not locule tissue of fruit, including fruit of the rin and nor mutants. It increased in amount in the pericarp tissues from an early stage to the mature green stage, clearly prior to any appreciable accumulation of the PG2 subunit. The -subunit polypeptide was not detected in stem or leaf tissues. A PG2-specific antiserum was used to study the interaction of PG2 with the isolated -subunit. The PG2 isoform was bound to the -subunit over a wide range of salt concentrations and pH; the interaction was independent of the presence of reducing agents. It is concluded that strong non-covalent forces are involved in the interaction. The results are consistent with a model in which the -subunit is positioned in the cell wall structure and provides a specific binding site for the active PG2 subunit when this is synthesised during ripening.Abbreviations B breaker - MG mature green - M_r relative molecular mass - nor non-ripening mutant - PAGE polyacrylamide gel electrophoresis - PG polygalacturonase - rin ripening inhibitor mutant - SDS sodium dodecyl sulphate     

Amphicarpum purshii and the “pessimistic strategy” in amphicarpic annuals with subterranean fruit

Summary Amphicarpum purshii Kunth is an annual amphicarpic grass of recently disturbed sandy areas in the Coastal Plain of eastern North America, producing small aerial and larger subterranean seeds. At study sites on the eastern edge of the Pine Barrens of New Jersey, the temporal aspects of reproductive allocation were investigated through biweekly whole-plant field harvests of 25 plants each throughout the 1980 growing season, and the survivorship of plants arising from aerial and subterranean seeds was monitored in a total of 10 high and 10 low density quadrats at two sites differeing in soil moisture. Plants arising from subterranean propagules began to allocate early (July 16) considerable energy to subterranean reproduction (ca 40% of whole-plant dry weight by the last half of August); allocation to aerial culms and flowers did not begin until mid-August and never exceeded 3% on a population basis. Plants arising from aerial propagules (aerial plants) produced only subterranean flowers, and these flowers first appeared 2 wk later than they did on subterranean plants. Survivorship was greater for the subterranean seedlings at both the dry and wet sites and at low and high densities, and aerial plants showed significantly less total growth and seed production than subterranean plants. The pessimistic strategy (early allocation of energy to large subterranean propagules) in the annual Amphicarpum has its selective basis in the relative vigor, survival, and timing and amount of reproduction of the two types of seedlings, and appears comparable to the allocation strategies of at least eight other amphicarpic annuals in at least five other families of plants.     

Cytochemical and immunocytochemical localization of Na,K-ATPase α subunit isoenzymes in the rat heart

In order to understand the functional significance of Na,K-ATPase subunits as well as their isoenzymes, a precise subcellular localization of these in the myocyte is a crucial prerequisite. Cytochemical, immunofluorescence, preembedding immunogold and horse radish peroxidasediaminobenzidine methods, demonstrated ₁ isoenzyme immunoreactivity on the sarcolemma, T-tubules and the subsarcolemmal cisterns of the adult cardiac myocytes. Cytochemically, ouabain resistant Na,K-ATPase precipitate was localized only in the subsarcolemmal cisterns and junctional sarcoplasmic reticulum. For ₂ isoenzyme, immunoreactivity was demonstrated on the sarcolemma as well as in all areas of the myocytes in particularly a close proximation to the sarcoplasmic reticulum and microsomes. For ₃ isoenzyme, only a weak insignificant signal was noted on the sarcolemma, intercalated disc and sarcoplasm. It is suggested that cytochemical ouabain resistant precipitate present in subsarcolemmal cisterns and junctional sarcoplasmic reticulum represent ₁ isoenzyme of Na,K-ATPase. A differential as well as unique localization of subunit isoenzymes of Na,K-ATPase in specific structures of cardiac myocytes may suggest importance in physiological function at these sites.     

Phosphates of the naphthol AS series in the quantitative determination of alkaline and acid phosphatase activities “in situ” studied in polyacrylamide membrane model systems and by cytospectrophotometry

Summary Histochemical media for the demonstration of alkaline and acid phosphatases using phosphates of naphthol AS series as the substrates and various diazonium salts as the couplers were tested in the capability of reflecting various levels of enzyme activities.Polyacrylamide membranes with incorporated enzymes (various concentrations of purified enzymes as well as of sonicated leucocytes, macrophages and of sonicated homogenates of various organs) were used as model systems in which the activity was estimated both with biochemical and with histochemical methods. Parallel experiments were performed in sedimentation chamber preparations of guinea-pig leucocytes and macrophages in which the activity was demonstrated with the same media as in polyacrylamide films. The quantitative measurements were performed in a cytospectrophotometer using the two-wavelength method.Increasing the substrate concentration which in standard histochemical media has been 1/8 mg per ml more azo-dye is produced in the reactions for both phosphatases. If the substrate concentration is higher than 1/2 mg per ml the standard concentration of the diazonium salt (1 mg per ml) becomes insufficient for an effective capturing of the released naphthol AS in the reaction for alkaline phosphatase. Due to a very high inhibitoty effect in the case of most commercially available diazonium salts the increase of their concentration annules the beneficial action of an increased substrate concentration on the azo-dye production. 4-amino-diphenylamine diazonium sulfate has an exceptional position because it was not inhibitory even in the concentration of 4 mg/ml.In the case of acid phosphatase the higher substrate concentration was incompatible with the use of Past Red Violet LB. Hexazo-p-rosanilin was an efficient and the most chromogenic coupler used in simultaneous as well as in postincubation coupling. With the latter localization is possible on the cellular (not subcellular) level.More chromogenic combinations are generally better for the cytospectrophotometrical measurement. The shape of extinction curves of azo-dyes produced with combinations studied was similar in models and in smears. In many combinations it was dependent on the presence of lipoproteins. A too steep decline of some curves prevented the use of some combinations in alkaline phosphatase determination with the two-wavelength method, even if they are very good in the qualitative studies and might be suitable for scanning cytospectrophotometry. p]The shape of extinction curves of azo-dyes produced in the reaction for acid phosphatase using hexazo-p-rosanilin as the coupling agent was independent of the presence of lipoproteins.The curves of azo-dyes produced in simultaneous coupling are not exactly the same as the curves obtained by postincubation coupling.In receipt of a fellowship of Netherlands Organization for the Advancement of Pure Research (Z.W.O.). Abbreviations used: AN-naphthol AS-AN phosphate; AS-naphthol AS-phosphate; B-Fast Blue B salt; BB-Fast Blue BB salt; BI-naphthol AS-BI phosphate; CL-naphthol AS-CL phosphate; DS-diazonium salt; GR-naphthol AS-GR phosphate; HP-hevazo-p-rosanilin; LB-Fast Red Violet LB salt; MX-naphthol AS-MX phosphate; S-substrate; TR-naphthol AS-TR phosphate (in the first half of the abbreviation), Fast Red TR salt (in the second half of the abbreviation); VB-4-amino-diphenylamine diazonium sulfate.     

D-amino acid oxidase fromTrigonopsis variabilis: Comparison of enzyme specificity “in vivo” and “in vitro”

Summary A method is described for permeabilization of intact cells of the yeastTrigonopsis variabilis with respect toin vivo measuring D-amino acid oxidase activity. The kinetic results so obtained differ from those obtained with the purified enzyme, pointing to the advantage of using the purified enzyme or the permeabilized cells in the oxidative deamination of different D-amino acids.     

Changing distribution patterns of α-glucosidase isoenzymes in extracts of antennal segments and in the haemolymph of the cotton stainer Dysdercus intermedius dist.

• 1.1. The α-glucosidase (E.C. 3.2.1.20) activities in extracts of the 2nd and 4th antennal segments (AS) and in the haemolymph of the cotton stainer were quantitatively examined by use of trehalose, p-nitrophenyl-α-glucoside, sucrose, turanose, maltose, isomaltose and palatinose as substrates.
• 2.2. Three peaks of activity were isolated by gel filtration from the extracts of both AS, but only two peaks from the haemolymph.
• 3.3. The peaks III of both AS differ in their substrate specificities from those of the peaks I and II.
• 4.4. It was evident from experiments, which used fractional extraction and variation of pH in addition to gel filtration that more than a single enzyme contributed to the formation of each peak.
• 5.5. Results are discussed with reference to the hypothesized presence of sugar reception processes in insect gustatory cells.

     

Kinetic and in silico analysis of thiazolidin-based inhibitors of α-carbonic anhydrase isoenzymes

Carbonic anhydrases (CAs, EC 4.2.1.1) are inhibited by sulfonamides, inorganic anions, phenols, salicylic acid derivatives (acting as drug or prodrugs). A novel class of CA inhibitors (CAIs), interacting with the CA isozymes I and II (cytosolic) in a different manner, is reported here. Kinetic measurements allowed us to identify thiazolidin-based compounds as submicromolar-low micromolar inhibitors of these two CA isozymes. Molecular docking studies of a set of such inhibitors within CA I and II active site allowed us to understand the inhibition mechanism. This new class of inhibitors bind differently compared to other classes of inhibitors known to date: they were found between the phenol-binding site, filling thus the middle of the enzyme cavity.     

Identity of the active site flavin-peptide fragments from the human “A”-form and the bovine “B”-form of monoamine oxidase

The monoamine oxidase present in the mitochondria of human placenta was verified to be the clorygyline sensitive or A form. This property was not abolished following extensive phospholipase treatment and Triton X-100 extraction. Proteolytic digestion of the partially purified enzyme using trypsin and chymotrypsin and isolation of a peptide containing the covalently linked flavin coenzyme permitted determination of the amino acid composition and sequence of the flavin region of the catalytic site of the enzyme. The structure of the flavin peptide was found to be identical to that of the bovine liver enzyme which is clorgyline insensitive, hence, the B form. The flavin peptide segment of mitochondrial monoamine oxidase is thus conserved between the two forms and among mammalian species.     

Effect of ploidy levels on the activities of Δ1-pyrroline-5-carboxylate synthetase,superoxide dismutase and peroxidase in Cenchrus species grown under water stress

The effects of ploidy levels on the activities of Δ¹-pyrroline-5-carboxylate synthetase (P5CS; EC not assigned), superoxide dismutase (SOD; EC 1.15.1.1) and guaiacol peroxidase (POX; EC 1.11.1.7), as well as malondialdehyde (MDA) and proline contents were studied in two months old plants of Cenchrus species. The Cenchrus species represent three ploidy levels: diploid, tetraploid, hexaploid and two life spans: annual and perennial. Plants were subjected to water stress for 2, 4, 6 and 8 d by withholding water under glasshouse conditions. Although the levels of proline increased with the magnitude of water stress, the P5CS activity did not show a corresponding increase in all species. Peroxidase and superoxide dismutase activities showed an increase or steady state in the early phase of drought and then declined with the further increase in the magnitude of water stress, indicating differing behaviors of species towards drought tolerance. Under drought, diploid Cenchrus species had a higher POX activity, MDA accumulation and lower proline content than tetraploid species. Lower POX and higher P5CS activities and proline contents, however, were observed in hexaploid and tetraploid species. Taken together, our findings suggest that diploid species have a less efficient antioxidant system to scavenge reactive oxygen species than tetra and hexaploid Cenchrus. This may result in a corresponding variability in growth and persistence under natural grasslands. The study also paves the way for investigations on the molecular events associated with drought in Cenchrus species differing in ploidy and life span.     

Characterization and expression of β-1,3-glucanase genes in peach

Beta-1,3-glucanase is one of the pathogenesis-related (PR) proteins involved in plant defense responses. A peach beta-1,3-glucanase gene, designated PpGns1, has been isolated and characterized. The deduced amino acid sequence of the product of PpGns indicates that it is a basic isoform (pI 9.8), and contains a putative signal peptide of 38 amino acids but has no C-terminal extension. Amino acid sequence comparisons revealed that PpGns1 is 69% and 67% identical to citrus and soybean beta-1,3-glucanases, respectively. Southern analysis of total genomic DNA also indicates that at least three genes for beta-1,3-glucanases exist in peach, forming a small gene family. Characterization of four additional clones by PCR has identified a second beta-1,3-glucanase gene, PpGns2. PpGns2 has been partially sequenced, and when compared to PpGns1, it shows high sequence homology, 96% and 99% nucleotide identity in the first and (partial) second exons, respectively. The deduced partial sequence of the PpGns2 product displays only two differences from PpGns1 in the signal peptide and one in the (partial) mature protein (141 amino acids). The 5'-flanking promoter regions of these two genes share 90% identity in nucleotide sequences interrupted by five major gaps (4-109 nt long). The promoter region contains various sequences similar to cis-regulatory elements present in different stress-induced plant genes. In leaves and stems of peach shoot cultures grown in vitro, PpGns1 is induced within 12 h after exposure to a culture filtrate of Xanthomonas campestris pv. pruni or ethephon. However, it is not induced following treatment with mercuric chloride.