In vivo demonstration of acid phosphatase activity in the rhizosphere of soil-grown plants

For in vivo demonstration of acid phosphatase activity in the rhizosphere of soil-grown plants filter papers were treated with a mixture of 1-naphthyl phosphate as substrate and the diazonium salt Fast Red TR as an indicator. After enzymatic hydrolysis, 1-naphthol forms a red complex with Fast Red TR. This method was applied to 8-day old maize plants and 3-year old Norway spruce plants growing in rhizoboxes in soil under non-sterile conditions. The treated filter paper is placed at the surface of roots and soil and acid phosphatase activity is visualized as a red-coloured root print on the filter paper. The method can be used as a qualitative analysis of acid phosphatase in the rhizosphere. It also allows a rough estimate of phosphatase activity in different root zones.     

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.     

Azoindoxylverfahren zum Hydrolasennachweis

Zusammenfassung An frischen, gefriergetrockneten und Schnitten von aldehyd-fixierten Rattengeweben werden 13 Diazoniumsalze als Simultankuppler zum Nachweis saurer, neutraler und alkalischer Hydrolasen mit Azoindoxylverfahren geprüft. Hexazotiertes Neufuchsin und/oder Fast Blue B sind die Diazoniumsalze der Wahl zur Lokalisation von saurer -Galactosidase, Neuraminidase, -N-Acetylglucosaminidase, saurer Phosphatase und unspezifischer Esterase gefolgt von Hexazonium-p-rosanilin. Fast Blue VB, BB und RR sowie Fast Violet B eignen sich zur Untersuchung von Lactase und alkalischer Phosphatase; Fast Garnet GBC kann zur Lokalisation von saurer -Galactosidase, Glucosaminidase und Lactase, Fast Red B, RC, RL und TR sowie Black K nur für Lactase-Studien verwandt werden. Durchschnittlich reichen 0,01–0,02 ml instabiles Diazoniumsalz und 0,3–1 mg stabiles Diazoniumsalz/ml zur korrekten Lokalisation dieser Hydrolasen aus. Im einzelnen hängt die Kupplerkonzentration von der Enzymaktivität und vom untersuchten Organ ab. Gefriergetrocknete Kryostatschnitte liefern unabhängig vom Kuppler die besten Resultate bei Untersuchung von Lactase und alkalischer Phosphatase; Schnitte von form- oder glutaraldehyd-fixierten Organen sind beim Nachweis der restlichen Hydrolasen überlegen. Eine Ausnahme macht die Untersuchung der sauren -Galactosidase und Glucosaminidase mit Fast Garnet GBC; dann werden die besten Ergebnisse nach Gefriertrocknung erzielt.Frische Kryostatschnitte sind zur Darstellung der Lactase mit hexazotiertem Neufuchsin oder p-Rosanilin und der alkalischen Phosphatase mit Fast Blue VB und BB sowie Violet B geeignet; die Gesamtaktivität der sauren, neutralen und alkalischen Hydrolasen kann mit semipermeablen Membranen und den stabilen sowie instabilen Diazoniumsalzen der Wahl untersucht werden.Ausreichende Osmierung der Azoindoxylfarbstoffe ist nur möglich, wenn Hexazonium-p-rosanilin als Kupplungsreagens benutzt wird; ohne Vorbehandlung extrahieren Äthanol, Isopropanol und Xylol alle Azoindoxyle.7 Inkubationsmedien werden zum lichtmikroskopisch-histochemischen Nachweis von Glykosidasen, Esterasen und Phosphatasen mit Azoindoxylmethoden angegeben und typische Anwendungsbeispiele genannt. Zur Gefriertrocknung von Kryostatschnitten mit dem Edwards-Pearse Gewebetrockner EPD 3 wird ein modifiziertes Verfahren beschrieben.

---

Azoindoxyl methods for the investigation of hydrolasesIV. Suitability of various diazonium salts

Summary Using fresh frozen, freeze-dried or cryostate sections from aldehyde fixed rat tissues 13 diazonium salts were tested as simultaneous coupling reagents for the localization of acid, neutral and alkaline hydrolases with azo indoxyl methods. Hexazotized new fuchsine and/or Fast blue B are the diazonium salts of choice for the demonstration of acid -galactosidase, neuraminidase, -N-acetylglucosaminidase, acid phosphatase, and non-specific esterase followed by hexazotized p-rosaniline. Fast blue VB, BB and RR and Fast violet B are recommended for the investigation of alkaline phosphatase and lactase, Fast garnet GBC for acid -galactosidase, glucosaminidase and lactase. Fast red B, RC, RL and TR and Fast black K can only be employed for lactase studies. The exact concentration of the coupling reagent depends on the activity of the enzyme and the organ investigated. On the average 0.01–0.02 ml unstable diazonium salt/ml and 0.3–1 mg stable diazonium salt/ml are sufficient for the correct localization of these hydrolases. Freeze-dried cryostat sections yield the best results in the demonstration of lactase and alkaline phosphatase independent on the coupling reagent used. Sections from formaldehyde or glutaraldehyde fixed organs are superior for the localization of the other hydrolases; an exception is the investigation of acid -galactosidase and glucosaminidase with Fast garnet GBC. Then, excellent results are obtained also with freeze-dried material.Fresh frozen sections are suitable for the localization of lactase with hexazotized new fuchsine or p-rosaniline and of alkaline phosphatase with Fast blue VB and BB or violet B. The total activity of acid, neutral and alkaline hydrolases can be investigated using semipermeable membranes in combination with all unstable and stable diazonium salts of choice.Reliable osmification of the azoindoxyl dye is only possible if hexazotized p-rosaniline is employed for coupling; without further posttreatment all azoindoxyl dyes are extracted by ethanol, isopropanol or xylol.7 incubation media are given for the demonstration of hydrolases with azoindoxyl methods at the level of light microscopy for routine studies and typical examples for the application of these methods are presented. A modified procedure is described for the freeze-drying of cryostat sections with the Edwards-Pearse tissue dryer EPD 3.

---

---

Mit Unterstützung durch die Deutsche Forschungsgemeinschaft (SFB 105)     

Azoindoxyl methods for the investigation of hydrolases. IV. Suitability of various diazonium salts (author's transl)]

Using fresh frozen, freeze-dried or cryostate sections from aldehyde fixed rat tissues 13 diazonium salts were tested as simultaneous coupling reagents for the localization of acid, neutral and alkaline hydrolases with azo indoxyl methods. Hexazotized new fuchsine and/or Fast blue B are the diazonium salts of choice for the demonstration of acid beta-galactosidase, neuraminidase, beta-N-acetylglucosaminidase, acid phosphatase, and non-specific esterase followed by hexazotized p-rosaniline. Fast blue VB, BB and RR and Fast violet B are recommended for the investigation of alkaline phosphatase and lactase, Fast garnet GBC for acid beta-galactosidase, glucosaminidase and lactase. Fast red B, RC, RL and TR and Fast black K can only be employed for lactase studies. The exact concentration of the coupling reagent depends on the activity of the enzyme and the organ imvestigated. On the average 0.01-0.02 ml unstable diazonium salt/ml and 0.3--1 microgram stable diazonium salt/ml are sufficient for the correct localization of these hydrolases. Freeze-dried cryostat sections yield the best results in the demonstration of lactase and alkaline phosphatase independent on the coupling reagent used. Sections from formaldehyde or glutaraldehyde fixed organs are superior for the localization of the other hydrolases; an exception is the investigation of acid beta-galactosidase and glucosaminidase with Fast garnet GBC. Then, excellent results are obtained also with freeze-dried material. Fresh frozen sections are suitable for the localization of lactase with hexazotized new fuchsine or p-rosaniline and of alkaline phosphatase with Fast blue VB and BB or violet B. The total activity of acid, neutral and alkaline hydrolases can be investigated using semipermeable membranes in combination with all unstable and stable diazonium salts of choice. Reliable osmification of the azoindoxyl dye is only possible if hexazotized p-rosaniline is employed for coupling; without further posttreatment all azoindoxyl dyes are extracted by ethanol, isopropanol or xylol. 7 incubation media are given for the demonstration of hydrolases with azoindoxyl methods at the level of light microscopy for routine studies and typical examples for the application of these methods are presented. A modified procedure is described for the freeze-drying of cryostat sections with the Edwards-Pearse tissue dryer EPD3.     

A quantitative histochemical study of dipeptidylpeptidase IV (DPP IV)

Summary In order to elucidate the possibility of a quantitative study of dipeptidylpeptidase IV (DPP IV) in cells and cell compartments of tissue sections kinetic investigations were performed with biochemical fluorometric and cytophotometric (microdensitometric) methods in the jejunum, kidney and liver of adult rats; in addition, two approaches of microdensitometric measuring (endpoint and continuous cytophotometry) were compared. Biochemically, Gly-Pro-4-methoxy-2-naphthylamine (MNA) is the best substrate compared with the 1-naphthylamine and 2-naphthylamine due to its high hydrolysis rate, the low Michaelis constant (K_m) and the relative high fluorescence of MNA. Its pH optimum is between 8.5 and 9. The hydrolysis rates delivered by cacodylate, phosphate and Tris HCl are similar and always higher than with other buffers. The maximal reaction velocity is reached with 3 mM Gly-Pro-MNA. The hydrolysis is inhibited in the presence of phenantroline, diisopronyl fluorphosphate, formaldehyde and diazonium salts; furthermore, formaldehyde and diazonium salts increase the Km of DPP IV. Fast Blue B pure (FBB) is the simultaneous coupling reagent of choice. End-point (plug and scanning procedure) and continuous microdensitometry with Gly-Pro-MNA and FBB in the intestinal and renal brush border and in renal glomerula have to be carried out at suboptimal pH (7.5). However, the optimal substrate concentrations are identical and the overall relation between the activity of DPP IV in the jenum, liver and kidney are similar in the quantitative histochemical and biochemical system. A linear relationship between the quantity of azo-dye and section thickness or incubation time exists between 4 m and 10 m and during the first 6 min of incubation respectively. Among different plotting procedures the best-fit method delivers the most reliable results. — The microdensitometric data in the small intestine show that K_m and V_max differ at different positions of the villi and may represent parameters for the maturation process of the enterocytes; in the kidney microdensitometry reveals different DPP IV activities in the different parts of the nephron. Both, the findings for the villi and the nephron cannot be obtained by biochemical methods.Supported by Deutsche Forschungsgemeinschaft (GU 184/1)Supported in part by Deutsche Forschungsgemeinschaft (SFB 105)     

Affinity of guanosine derivatives for polycytidylate revisited

Evidence is presented for complexation of guanosine 5-monophosphate 2-methylimidazolide (2-MeImpG) with polycytidylate (poly(C)) at pH 8.0 and 23°C in the presence of 1.0 M NaCl and 0.2 M MgCl₂ in water. The association of 2-McImpG with poly(C) was investigated using UV-vis spectroscopy as well as by monitoring the kinetics of the nucleophilic substitution reaction of the imidazole moiety by amines. The results of both methods are consistent with moderately strong poly(C) · 2-McImpG complexation and the spectrophotometric measurements allowed the construction of a binding isotherm with a concentration of 2-McImpG equal to 5.55 ± 0.15 mM at half occupancy. UV spectroscopy was employed to establish the binding of other guanosine derivatives on poly(C). These derivatives are guanosine 5-monophosphate (5GMP), guanosine 5monophosphate imidazolide (ImpG), and guanosine 5monophosphate morpholidate (morpG). Within experimental error these guanosine derivatives exhibit the same affinity for poly(C) as 2-McImpG.     

Cytochemical evidence for the leakage of acid phosphatase through ultrastructurally intact lysosomal membranes

Synopsis Fixation under improper conditions ofin vitro cultivated cells results in an extensive diffusion of the lysosomal enzyme acid phosphatase because of the influence of a low effective osmotic pressure. In the present investigation, advantage was taken of this predictable diffusion in order to establish whether or not leakage of acid phosphatase could take place through ultrastructurally intact lysosomal membranes.In order to reveal small holes in the lysosomal membranes, secondary lysosomes were labelled with thorium dioxide particles, which were presumed to appear free in the cell sap if ruptures in the membranes larger than about Ioo Å were created.The experiments revealed that following the fixation ofin vitro cultivated human glia cells under improper conditions, mitochondria and ground cytoplasm show considerable swelling artifacts, while secondary lysosomes appear to be essentially unaffected. The lysosomes, nevertheless, apparently lost most of their content of acid phosphatase, as judged from enzyme cytochemical studies. These findings indicate that leakage of acid phosphatase from ultrastructurally intact lysosomes is possible.     

Cloning, expression and characterization of a UDP-galactose 4-epimerase from Escherichia coli

The gene galE encoding UDP-galactose 4-epimerase was cloned into E. coli BL21(DE3) from the chromosomal DNA of E. coli strain K-12. High expression of the soluble recombinant epimerase was achieved in the cell lysate. In order to evaluate the use of this epimerase in enzymatic synthesis of important -Gal epitopes (oligosaccharides with a terminal Gal1,3Gal sequence), a new radioactivity assay (1,3-galactosyltransferase coupled assay) was established to characterize its activity in producing UDP-galactose from UDP-glucose. Approximately 2700 units (100 mg) enzyme with a specific activity of 27 U mg^–1 protein could be obtained from one liter of bacterial culture. The epimerase was active in a wide pH range with an optimum at pH 7.0. This expression system established a viable route to the enzymatic production of -Gal oligosaccharides to support xenotransplantation research.     

A test of the binary chloroplast membrane hypothesis by using a nonpenetrating chemical probe,p-(diazonium)-benzenesulfonic acid

Summary Spinach chloroplasts were exposed to³⁵S-labeledp-(diazonium)-benzenesulfonic acid (DABS), a water soluble compound which does not penetrate lipophilie regions of membranes, and which is highly reactive toward amino acid functionagroups such as -amino, sulfhydryl, histidine, and tyrosine groups. Amino groups inl lipids can also form similar, stable covalent bonds by diazo coupling. Both chloroplast lipids and proteins were labeled with DABS, the total binding being about 1 DABS per 10 chlorophylls, depending on the reaction conditions.After diazo coupling and subsequent digitonin fractionation into photosystems I and II enriched fractions, it was observed that PS-I was more highly labeled than PS-III usually by a factor of 10 to 24 times (on a per chlorophyll basis). After digitonin isolation, however, the PS-II portion bound an amount of DABS similar to the PS-I binding, We interpret these data as consistent with the binary membrane hypothesis (Arntzen. Dilley and Crane (1969),J. Cell Biol. 43:16), which visualizes PS-I on the externa, half of a 90 Å grana membrane, and PS-II occurring on the interior half of thel membrane. The alternative explanation that PS-II and PS-I are arranged as a mosaic, and that the low DABS binding in PS-II is caused by burial of the diazo reactive groups in the interior of the proteins (and only exposed through the denaturing effect of digitonin) is not directly ruled out. However, this alternative is not consistent with the facts that: (a) most of the membrane proteins in PS-I and PS-II are identical in electrophoretic properties and therefore probably have similar overall structures; and (b) digitonin does not lead to appreciable denaturation of proteins, evidenced by the retention of PS-II electron transport activity.     

Rooting apple cultivars in vitro: Interactions among light,temperature, phloroglucinol and auxin

Delicious apple (Malus domestica Borkh.) and several of its strains, which have been difficult to root in vitro, were successfully propagated with rooting percentages up to 100%. The combination of treatments used to achieve this result included placing the shoots on rooting medium in the dark at 30°C for the first week of the rooting stage, then moving them to a regime of 16 hr light-8 hr dark at 25°C. The rooting medium contained half strength Murashige and Skoog salts plus 1.2 M thiamine HCl, 0.56 mM myo-inositol, 1 mM phloroglucinol (PG), 1.4 M indolebutyric acid (IBA), 1.3 M gibberellic acid (GA₃), 87.6 mM sucrose, and 7 g l^–1 Difco Bacto agar. Dark treatment applied during the proliferation stage (etiolation) was less effective than one applied at the beginning of the rooting stage. The optimum length of dark treatment during rooting was 4 to 7 days. Increasing the temperature from 25°C to 30°C improved rooting of Delicious, Royal Red Delicious, and Vermont Spur Delicious in the absence of PG but generally had less effect in the presence of PG. Further increase in temperature to 35°C stimulated rooting of Royal Red Delicious but reduced rooting of Vermont Spur Delicious. Transfer of the cuttings to auxin-free medium after 1 week had no effect on percentage rooting and increased the number of roots per cutting for only 1 of 4 cultivars tested and then only in the presence of PG. In general PG stimulated rooting of Delicious and its strains, but had no effect on Golden Delicious.     

5′-Nucleotidase

Summary Determinations of pH activity curves and of Michaelis constants of 5-nucleotidases in organs of rat and mouse indicate the heterogenity of the enzyme in these tissues. Electrophoretic analyses of homogenates and cell component fractions reveal the presence of 5-nucleotidase isoenzymes in the investigated tissues. At the acid as well as at the neutral pH five isoenzymes were found. In addition three alkaline phosphatases were found in the rat; in the mouse four alkaline phosphatase isoenzymes could be demonstrated. The different combinations of 5-nucleotidase isoenzymes in the investigated tissues possibly indicate different functions of the isoenzymes. A discussion is given of the correlations between the electrophoretic results and the histochemical findings.     

Transfer of Ogu cytoplasmic male sterility to Brassica juncea and improvement of the male sterile line through somatic cell fusion

Male sterility conferred by ogu cytoplasm of Raphanus sativus has been transferred to Brassica juncea cv RLM 198 from male-sterile B. napus through repeated backcrossing and selection. The male-sterile B. juncea is, however, highly chlorotic and late. It has low female (seed) fertility and small contorted pods. To rectify these defects, protoplasts of the male sterile were fused with normal RLM 198 (green, self fertile). Four dark green, completely male-sterile plants were obtained and identified as putative cybrids. All the plants were backcrossed three times with RLM 198. Mitochondrial and chloroplast DNA analysis of backcross progeny confirmed hybridity of the cytoplasm. The restriction pattern of the chloroplast DNA of progeny plants of three cybrids (Og 1, Og 2, Og 3) was similar to that of the green self-fertile RLM 198 and indicated that the correction of chlorosis resulted from chloroplast substitution. The chloroplast DNA of the lone progeny plant of the fourth cybrid (Og 10) could not be analyzed because the plant was stunted and had only a few leaves. When total cellular DNA was probed with mitochondrial probes coxI and atpA it was found that the cybrids had recombinant mitochondria. The chlorosis-corrected plants were early flowering and had vastly improved seed fertility.     

The ability of acidic pH,growth inhibitors,and glucose to increase the proton motive force and energy spilling of amino acid-fermenting <Emphasis Type="Italic">Clostridium sporogenes</Emphasis> MD1 cultures

Clostridium sporogenes MD1 grew rapidly with peptides and amino acids as an energy source at pH 6.7. However, the proton motive force (p) was only –25 mV, and protonophores did not inhibit growth. When extracellular pH was decreased with HCl, the chemical gradient of protons (ZpH) and the electrical membrane potential () increased. The p was –125 mV at pH 4.7, even though growth was not observed. At pH 6.7, glucose addition did not cause an increase in growth rate, but increased to –70 mV. Protein synthesis inhibitors also significantly increased . Non-growing, arginine-energized cells had a of –80 mV at pH 6.7 or pH 4.7, but was not detected if the F₁F₀ ATPase was inhibited. Arginine-energized cells initiated growth if other amino acids were added at pH 6.7, and and ATP declined. At pH 4.7, ATP production remained high. However, growth could not be initiated, and neither nor the intracellular ATP concentration declined. Based on these results, it appears that C. sporogenes MD1 does not need a large p to grow, and p appears to serve as a mechanism of ATP dissipation or energy spilling.Mandatory disclaimer: Proprietary or brand names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by the USDA implies no approval of the product, and exclusion of others that may be suitable.     

Effect of mutant host RNA polymerase on the bifunctional activities of P22 gene c1.

Summary A phage has been isolated which specifically transduces the Escherichia coli pheS and pheT genes coding for the and subunits of the phenylalanyl-tRNA synthetase (PRS). This phage transduces with high frequency (i) several temperaturesensitive PRS mutants to thermoresistance and (ii) a p-fluorophenylalanine resistant PRS mutant to sensitivity against this amino-acid analog. The in vitro PRS activities of such lysogens suggest that the and subunits coded by the transducing phage complement the mutant host PRS-subunits in vivo by means of formation of hybrid enzymes.The transducing phages were also used to infect UV light irradiated cells. The SDS-gel electrophoretic analysis of the proteins synthesized in such cells revealed that the phage codes at least for four different E. coli proteins. Two proteins with molecular weights of 94,000 and 38,000 daltons cross-reacted with an anti PRS serum and were thus identified as the and subunits of PRS, respectively. A third protein with w molecular weight of 22,000 daltons is identical with the ribosomal initiation factor IF3 (Springer et al., 1977b). The other protein (M_r 78,000) is still unidentified.     

Effects of osmotic stabilizers on the activities of mycolytic enzymes used in fungal protoplast liberation

Summary Assay methods for chitinase, -glucanase and -glucanase in the presence of the osmotic stabilizers used in fungal protoplast liberation were developed. Chitinase activity with an inorganic osmotic stabilizer system was in the order of NO ₃ ^– , Cl^–>SO ₄ ^2– >PO ₄ ^3– and Na⁺, K⁺>Ca²⁺, Mg²⁺. Monovalent anion salts with monovalent cations improved chitinase activity, whereas divalent and trivalent anion and cation salts caused appreciable inhibition; phosphate salts induced very serious inhibition. These phenomena suggest that a suitable electrical state is required for optimal chitinase activity. MgSO₄, KCl and NH₄Cl were equally efficient as stabilizers for protoplast liberation, although they had different effects on chitinase activity. -glucanase was inhibited more by sucrose than by mannitol and sorbitol; -glucanase was relatively stable to both organic and inorganic osmotic stabilizers. As chitin is the major component of the fungal cell wall, chitinase is thought to be more important for protoplast liberation than are -glucanase and -glucanase.

---

Resumen Se han desarrollado técnicas para la determinación de: quitinasa, -glucanasa y -glucanasa en presencia de estabilizadores osmóticos usados en la liberación de protoplastos fúngicos. La actividad quitinasa con estabilizadores inorgánicos siguió el orden: NO ₃ ^– , Cl^–>SO ₄ ^2– >PO ₄ ^3– Na⁺, K⁺>Ca²⁺, Mg²⁺. Los aniones monovalentes junto con los cationes monovalentes mejoraron la actividad quitinasa, mientras que tanto aniones como cationes, divalentes y trivalentes causaron una inhibición apreciable. Las sales de fosfato indujeron inhibiciones muy severas. Estos fenómenos sugieren que un estado eléctrico adecuado es necesario para una actividad quitinasa óptima. MgSO₄, KCl y NH₄Cl fueron igualmente eficientes como estabilizadores para la liberación de protoplastos, aunque tuvieran distintos efectos en la actividad quitinasa. La -glucanasa se inhibió más por sucrosa que por azucares-alcoholes; la -glucanasa se mantuvo relativamente estable frente a estabilizadores osmóticos tanto orgánicos como inorgánicos. Al ser la quitina el componente mayoritario de la pared celular de los hongos, se cree que la quitinasa es más importante que la -glucoanasa y la -glucanasa en lo que concierne a la liberación de protoplastos.

---

Résumé Des méthodes ont été mises au point pour mesurer les activités -et -glucanase en présence des stabilisateurs osmotiques utilisés pour la production de protoplastes. En présence d'un système minéral de stabilisation osmotique, l'activité chitinase est dans l'ordre NO ₃ ^– , Cl^–>SO ₄ ^2– >PO ₄ ^3– et Na⁺, K⁺>Ca²⁺, Mg²⁺. Les anions monovalents ainsi que les cations monovalents améliorent l'activité chitinase, tandis que les anions et les cations di- et tri-valents l'inhibent de façon appréciable; les phosphates sont fortement inhibiteurs. Ces phénomènes suggèrent que l'activité chitinase optimale exige un état électrique approprié. MgSO₄, KCl et NH₄Cl stabilisent de façon identique la formation des protoplastes, bien que leurs effets respectifs sur l'activité chitinase soient différents. L' -chitinase est inhibée par le saccharose davantage que par les sucres-alcools; la -glucanase est relativement stable en présence des stabilisateurs organiques ou minéraux. Comme la chitine est le constituant majeur des parois cellulaires fungiques, on considère que la chitinase est plus importante que la -et l' -glucanase pour la formation des protoplastes.

     

A self-fertile trigeneric hybrid,Triticum aestivum ×Agropyron michnoi ×Secale cereale

Trigeneric hybrids between the (Triticum aestivum ×Agropyron michnoi) F₁ (CM, 2n=5x=35; ABDPP) and two winter rye (Secale cereale L., 2n=2x=14; RR) cultivars, Wugong 774 and AR-132, were synthesized. Such trigeneric hybrids could be used to transfer resistance genes for powdery mildew from rye to CM and subsequently to common wheat and to identify (1) the effects of the P genome ofAgropyron on the self-fertility of the hybrids and (2) the differences in genetic background between rye cultivars with marked differences in pollinating habit. The trigeneric hybrids varied widely in morphology and showed a high level of resistance to such diseases as barley yellow dwarf virus (BYDV), stripe rust, leaf rust, stem rust, and powdery mildew. Selfed and many backcross derivatives were obtained from the trigeneric hybrids. The results indicated that rye cvs Wugong 774 and AR132 arose from different gene pools and that the P genome ofAgropyron carries gene(s) responsible for chromosome segregation, leading to functional gamete formation and self-fertility of the hybrids. The F₂ and BC₁ plants could be obtained in two ways — fusion of the unreduced gametes and the assumed apomixis of unreduced female gametes in the trigeneric hybrid plant II-4 — which indicates that this trigeneric hybrid may be a special genetic stock. Chromosome pairing in the trigeneric hybrids and ways of producing wheat/rye and wheat/Agropyron translocations are discussed.     

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.     

Synthesis of aspartame precursor by solid thermolysin in organic solvent

Summary The enzymatic synthesis of a peptide compound was carried out successfully in homogeneous organic solvent.Solid Thermolysin was found to catalyze the synthetic reaction of N-benzyloxycarbonyl-L-aspartyl-L-phenylalanine methyl ester (Z-APM; a precursor of sweetner Aspartame) from N-benzyloxycarbonyl-L-aspartic acid (Z-L-Asp) and L-phenylalanine methyl ester (L-PheOMe) in a 98 percent organic medium (ethylacetatebenzenemethanolwater=5029192). The dissolution of enzyme was not observed. The optimal pH shifted to acidic side by 1.0 pH unit, compared with that in aqueous medium. The enzymatic activity of solid thermolysin with an average size of 3.4×9.5 m was determined to be 0.18 moles-product/(mg-solid)·h under the initial concentrations of L-PheOMe of 0.1M and Z-L-Asp of 0.05M, and at pH 6.0 and 40°C.     

The challenge of sustainability: Is integrating environment and economy enough?

Conclusions Beyond Interdependence, along with each of the other books, appeared after publication of Our Common Future but before the Earth Summit in Rio. It was a time to be optimistic, and each book is optimistic, if cautiously so. Certainly the impact of the Brundtland Report has been enormous, perhaps not beyond the dreams of the members of the Commission, but certainly beyond their expectations. The impact of the Earth Summit has been much smaller, certainly well short of the dreams of those involved though perhaps within their expectations.If the Brundtland Report and the books reviewed in this essay show the potential, the Earth Summit shows the limits. What have been called the inner limits of interests, institutions, and politics turn out to be far more important than the outer limits of ecology and even economics. The next steps are going to be difficult, and books such as Beyond Interdependence are needed to indicate when, where and how to take such steps. There may eventually be a Mark II framework convention for climate change, forestry and biodiversity (p. 115–116), but along the way there will be many more national actions, bi- or tri-national agreements, and Montreal Protocols. In the words of MacNeill, Winsemius and Yakushiji (p. 117), A Grand Global Bargain could be the sum of 1,000 small bargains. Even if, in my view and the views of Goodland et al. and Meadows et al., Beyond Interdependence is inadequate to get us onto a fully sustainable path, there is little if anything in its recommendations that would be inappropriate in our search for that path.How can we go beyond integration of environment and economy? A number of authors have begun to make suggestions (e.g., Ekins, 1987; Brooks, 1991; Durning, 1992; Harrison, 1992). Certainly, one can agree with MacNeill, Winsemius and Yakushiji that we need not spend any more time defining sustainable development. (By 1990, there were already some 40 definitions in the literature.) The definition provided by the Brundtland Report is good enough for working purposes. What is needed now is not more discussion about sustainable development but more experimentation with it. We must try different development strategies, and keep trying until the best fit is found for particular times, particular places and particular peoples. Some policies will get us closer; others will turn out to be inefficient, destructive, or inequitable. But the experiments must continue until each community finds a set that works economically, ecologically and politically. In the words of Rabbi Tarphon, who lived nearly two millennia ago and contributed to the Talmud, It is not up to us to complete the task, but neither are we free to refrain from getting started on it.     

Extracellular amylolytic system of the yeast Lipomyces kononenkoae

Summary A strain of the yeast Lipomyces kononenkoae which converted starch into SCP with a high yield, produced three extracellular amylases which were purified from the culture fluid by Ficoll concentration, dialysis, isopropanol precipitation and DE-cellulose chromatography: an -amylase, a glucoamylase and a debranching transferase. The latter transferred -1,6-glucosyl units from panose to glucose forming maltose and appeared to have some debranching activity on amylopectin. The -amylase had the following properties: MW 38000 daltons; no effect of added calcium ions on activity; optimum temperature and pH for activity around 40°C and pH 5.5; H and S of heat inactivation 24360 cal mol^–1 and 29.2 cal deg^–1 mol^–1; range of pH stability pH 4–6.5; pI=7.1; final low molecular weight products of starch hydrolysis, maltose and glucose; K_m (40°C, pH 5.5) for starch 2.7 gl^–1, for maltotriose 109 gl^–1; uncompetitive inhibition by maltose with K_i (40°C, pH 5.5) 29.5 gl^–1. The glucoamylase had the following properties: MW 81500 daltons; optimum temperature and pH for activity around 50°C and pH 4.5: H and S of heat inactivation 20400 cal mol^–1 and 17.7 cal deg^–1 mol^–1; range of pH stability pH 4–6.5; pI=6.1; K_m (30°C, pH 4.5) for soluble starch 16.2 gl^–1, for maltose 0.36 gl^–1, for p-nitrophenyl--D-glucoside 0.35 gl^–1; competitive inhibition by glucose with K_i (30°C, pH 4.5) 4.7 gl^–1.