1,2,5-Trisubstituted 1,4-diazepine-3-one: A novel dipeptidomimetic molecular scaffold

The continuing effort to transform bioactive peptides into non-peptide peptidomimetics of therapeutic potential requires a diversity of tools such as molecular scaffolds, pseudopeptide modifications, and conformation mimetics. To this end, a novel polyfunctional monoheterocyclic system, 1,2,5-trisubstituted hexahydro-3-oxo-1H-1,4-diazepine ring (DAP), was designed. The linear precursor for the DAP was generated through a reductive alkylation step including a modified side chain and an -amino function of two amino acid derivatives. Structural analysis of model diastereomeric DAPs, employing ¹H and¹³ C NMR and computer simulation, revealed the conformational preferences of this system. The structural similarities to the 1,4-benzodiazepine, a common molecular scaffold for many non-peptidic peptidomimetic agents, and the pronounced dipeptidomimetic character of the DAP system offer a new powerful tool to medicinal chemists engaged in rational peptide-based drug design.     

Alley cropping with Senna siamea in South-western Nigeria: I. Recovery of 15N labeled urea by the alley cropping system

Improved cropping systems with in-situ production of organic matter require the input of additional inorganic N to maintain crop production in a sustainable way. For proper management of this fertilizer-N, it is necessary to quantify how the applied fertilizer N is used by the various components of the system and by the system as a whole. The fate of a single application of ¹⁵N labeled urea-N through the different components (crop, hedgerow, surface litter, soil profile up to 150 cm) of a Senna siamea alley cropping system, intercropped with maize in the first and cowpea in the second season, was followed for a period of 1.5 years (1994–1995), equivalent to 2 maize and 1 cowpea crop. Special attention was given to the role of the particulate organic matter (POM) in the cycling of urea-N through the soil organic matter (SOM). The maize crop recovered 26.5 and 1.7% of the applied urea-N at harvest in 1994 and 1995, respectively. The cowpea pods recovered only 0.7% of the applied urea-N at harvest. The highest proportion of applied urea-N recovered by the hedgerow occurred at 38 days after 1994 maize planting (DAP) (3.8%), while at later dates, recoveries of applied urea-N were always below 1%. This indicates that the Senna hedge is not a strong competitor for the applied urea-N during crop growth, i.e. while the Senna canopy is pruned at regular intervals. At 21 DAP, 12.7% of the applied urea-N was recovered in the surface litter and this value dropped significantly to 1.6% at 107 DAP and remained below 1% up to 480 DAP. The top 10 cm of soil contained 21% of the applied urea-N at 21 DAP and this value dropped to 9% at 480 DAP. Significantly more urea-N was recovered in the top 10 cm of soil than in the deeper soil layers at all sampling times. At 21 DAP, 11% of the applied urea-N was recovered in the 120–150 cm layer. This fast movement of urea-derived N to deep soil layers must have happened by preferential flow in macropores as the rainfall between urea application and the first sampling (74.2 mm) was not high enough to explain downward movement of N with the mobile water. Significant linear relationships between the proportion of urea-N in the different soil layers (excluding 0–10 cm) and the anion exchange capacity (AEC) and silt+clay content of the respective layers were found at 67, 107, 347 and 480 DAP. The total N content of the POM fraction increased significantly between 0 and 101 DAP from 127 to 171 mg N kg^–1 and decreased to 92 mg N kg^–1 at 480 DAP. The highest recovery of applied urea-N in the POM pool was measured at 101 DAP (3.6%) and this value decreased to 1.8% at 480 DAP. The total recovery of applied urea-N was 81% at 21 DAP, and decreased to values varying between 53 and 60% up from 38 to 347 DAP. At 480 DAP, the recovery decreased further to 47%. The fast movement of a substantial amount of urea-N may be responsible for this incomplete recovery, already at 21 DAP. Although the soil N status in the fertilized alley cropping system appears to be favourable for plant growth, this may be short-lived in the absence of further urea additions, as the soil-derived maize uptake in 1995 was already significantly lower than in 1994, and as the labile POM pool decreased significantly between the maize harvest in 1994 and 1995.     

Chemoenzymatic synthesis of multivalent neoglycoconjugates carrying the helminth glycan antigen LDNF

Several parasitic helminthes, such as the human parasite Schistosoma mansoni, express glycoconjugates that contain terminal GalNAcβ1-4(Fucα1-3)GlcNAcβ-R (LDNF) moieties. These LDNF glycans are dominant antigens of the parasite and are recognized by human dendritic cells via the C-type lectin DC-SIGN. To study the functional role of the LDNF antigen in interaction with the immune system, we have developed an easy chemoenzymatic method to synthesize multivalent neoglycoconjugates carrying defined amounts of LDNF antigens. An acceptor substrate providing a terminal N-acetylglucosamine was prepared by coupling a fluorescent hydrophobic aglycon, 2,6-diaminopyridine (DAP), to N,N′-diacetylchitobiose. By the subsequent action of recombinant Caenorhabditis elegans β1,4-N-acetylgalactosaminyltransferase and human α1,3-fucosyltransferase VI (FucT-VI), this substrate was converted to the LDNF antigen. We showed that human FucT-VI has a relatively high affinity for the unusual substrate GalNAcβ1-4GlcNAc (LDN), and this enzyme was used to produce micromolar amounts of LDNF–DAP. The synthesized LDNF–DAP was coupled to carrier protein via activation of the DAP moiety by diethyl squarate. By varying the molar glycan:protein ratio, neoglycoconjugates were constructed with defined amounts of LDNF, as was determined by MALDI-TOF analysis and ELISA using an anti-LDNF antibody.     

Alley cropping with Senna siamea in South-western Nigeria: II. Dry matter,total N,and urea-derived N dynamics of the Senna and maize roots

Crop and tree roots are crucial in the nutrient recycling hypotheses related to alley cropping systems. At the same time, they are the least understood components of these systems. The biomass, total N content and urea-derived N content of the Senna and maize roots in a Senna-maize alley cropping system were followed for a period of 1.5 years (1 maize-cowpea rotation followed by 1 maize season) to a depth of 90 cm, after the application of ¹⁵N labeled urea. The highest maize root biomass was found in the 0–10 cm layer and this biomass peaked at 38 and 67 days after planting the 1994 maize (DAP) between the maize rows (112 kg ha^–1, on average) and at 38, 67 and 107 DAP under the maize plants (4101 kg ha^–1, on average). Almost no maize roots were found below 60 cm at any sampling date. Senna root biomass decreased with time in all soil layers (from 512 to 68 kg ha^–1 for the 0–10 cm layer between 0 and 480 DAP). Below 10 cm, at least 62% of the total root biomass consisted of Senna roots and this value increased to 87% between 60 and 90 cm. Although these observations support the existence of a Senna root `safety net' between the alleys which could reduce nutrient leaching losses, the depth of such a net may be limited as the root biomass of the Senna trees in the 60–90 cm layer was below 100 kg ha^–1, equivalent to a root length density of only < 0.05 cm cm^–3. The proportion of maize root N derived from the applied urea (%Ndfu) decreased significantly with time (from 21% at 21 DAP to 8% at 107 DAP), while %Ndfu of the maize roots at the second harvest (480 DAP) was only 0.6%. The %Ndfu of the Senna roots never exceeded 4% at any depth or sampling time, but decreased less rapidly compared to the %Ndfu of the maize roots. The higher %Ndfu of the maize roots indicates that maize is more efficient in retrieving urea-derived N. The differences in dynamics of the %Ndfu also indicate that the turnover of N through the maize roots is much faster than the turnover of N through the Senna roots. The recovery of applied urea-N by the maize roots was highest in the top 0–10 cm of soil and never exceeded 0.4% (at 38 DAP) between the rows and 7.1% (at 67 DAP) under the rows. Total urea N recovery by the maize roots increased from 1.8 to 3.2% during the 1994 maize season, while the Senna roots never recovered more than 0.8% of the applied urea-N at any time during the experimental period. These values are low and signify that the roots of both plants will only marginally affect the total recovery of the applied urea-N. Measurement of the dynamics of the biomass and N content of the maize and Senna roots helps to explain the observed recovery of applied urea-N in the aboveground compartments of the alley cropping system.     

Rapid regeneration of <Emphasis Type="Italic">Phaseolus angustissimus</Emphasis> and <Emphasis Type="Italic">P. vulgaris</Emphasis> from very young zygotic embryos

A rapid regeneration protocol for proembryos of Phaseolus angustissimus as young as 1 day after pollination (DAP) involving pod culture for 1 week followed by embryo culture for 2 weeks and embryo germination for 1 or 2 weeks is provided. Optimization of the media was conducted with pods collected 3 DAP. The best pod culture medium was composed of basal medium [(Phillips and Collins 1979) salts with (Geerts et al. 2001) vitamins], 1000 mg l⁻¹ glutamine, 1000 mg l⁻¹ casein hydrolysate, 3% sucrose and 0.5% agar. Embryo culture medium consisted of basal medium with 500 mg l⁻¹ glutamine, 250 mg l⁻¹ casein hydrolysate, 1.9 μM ABA, 3% sucrose and 0.5% bacto-agar. Embryos developed into plantlets on germination medium containing basal medium with 0.25 μM BA, 3% sucrose and 0.7% bacto-agar. Fertile, normal plants were recovered from direct embryogenesis and from micrografted embryo-derived shoots. Embryos obtained from pods collected 3 DAP regenerated plantlets at a rate of 29.3%, while embryos from pods collected 2 DAP and 1 DAP regenerated at rates of 20.2 and 4%, respectively. A second accession of P. angustissimusregenerated at a rate of 26.2%. Using this 5-week protocol for P. vulgaris resulted in a plantlet regeneration rate of 12.5%.     

3D-QSAR studies of checkpoint kinase 1 inhibitors based on molecular docking and CoMFA

Three-dimensional quantitative structure–activity relationship (3D-QSAR) studies were performed on a series of substituted 1,4-dihydroindeno[1,2-c]pyrazoles inhibitors, using molecular docking and comparative molecular field analysis (CoMFA). The docking results from GOLD 3.0.1 provide a reliable conformational alignment scheme for the 3D-QSAR model. Based on the docking conformations and alignments, highly predictive CoMFA model was built with cross-validated q ² value of 0.534 and non-cross-validated partial least-squares analysis with the optimum components of six showed a conventional r ² value of 0.911. The predictive ability of this model was validated by the testing set with a conventional r ² value of 0.812. Based on the docking and CoMFA, we have identified some key features of the 1,4-dihydroindeno[1,2-c]pyrazoles derivatives that are responsible for checkpoint kinase 1 inhibitory activity. The analyses may be used to design more potent 1,4-dihydroindeno[1,2-c]pyrazoles derivatives and predict their activity prior to synthesis.     

Efficient production and partial characterization of aspartyl aminopeptidase from Aspergillus oryzae

Aims: Aspartyl aminopeptidase (DAP) has a high degree of substrate specificity, degrading only amino-terminal acidic amino acids from peptides. Therefore, attention is focused here on the efficient production of this enzyme by a recombinant Aspergillus oryzae and characterization of its biochemical properties. Methods and Results: The gene encoding DAP was overexpressed under a taka-amylase gene promoter, with His-tag linker in A. oryzae, during cultivation in a Co²⁺-containing medium. The enzyme was extracted from the mycelia and purified with immobilized nickel ion absorption chromatography using a buffer containing cobalt ion and imidazole. The active fraction was further purified with gel filtration chromatography. The resultant, electrophoretically pure enzyme displayed a molecular mass of 520 kDa. This enzyme displayed high reactivity towards peptide substrate rather than synthetic substrates. Conclusions: Recombinant A. oryzae DAP was purified to homogeneity with an increased specific activity, when cultivated in a Co²⁺-rich medium. Moreover, the use of suitable metal ions in microbial cultivation and purification processes may help in increasing the specific activity of other metalloproteases and their functional analysis. Significance and Impact of the Study: Recombinant DAP produced using a cobalt ion in culture media of A. oryzae and purification process allow high yield of the enzyme activity.     

Responses of morphological, physiological, and biochemical parameters in Euglena gracilis to 7-days exposure to two commonly used fertilizers DAP and urea

Diammonium phosphate (DAP) and urea are commonly used fertilizers throughout the world. The effects of these fertilizers on the freshwater flagellate Euglena gracilis was studied after 7 days of growth using morphological, physiological and biochemical parameters as end points. NOEC and EC₅₀ values for various parameters like cell density, motility, velocity, cell shape, gravitaxis, chlorophyll a, b and total carotenoids were calculated. NOEC and EC₅₀ values of DAP varied from 0.5 to 2.5 g L⁻¹ and 3.14 to 5.96 g L⁻¹, respectively, for different parameters. NOEC and EC₅₀ values for urea ranged from 5 to 25 g L⁻¹ and 28 to 44.05 g L⁻¹, respectively, for various parameters. Photosynthetic pigments were found to be more sensitive to both fertilizers as compared to other measured end points. The NOEC and EC₅₀ values obtained for DAP were much lower than those for urea; i.e., DAP showed a stronger inhibitory effect as compared to urea. Application of DAP resulted in an increased concentration of ammonia in Euglena cultures but urea did not. The stronger inhibitory effect of DAP is attributed to release of free ammonia in the culture due to DAP decompostion. No release of ammonia by urea occurred due to the absence of the enzyme urease in E. gracilis.     

LiaR‐independent pathways to daptomycin resistance in Enterococcus faecalis reveal a multilayer defense against cell envelope antibiotics

The lipopeptide antibiotic daptomycin (DAP) is a key drug against serious enterococcal infections, but the emergence of resistance in the clinical setting is a major concern. The LiaFSR system plays a prominent role in the development of DAP resistance (DAP‐R) in enterococci, and blocking this stress response system has been proposed as a novel therapeutic strategy. In this work, we identify LiaR‐independent pathways in Enterococcus faecalis that regulate cell membrane adaptation in response to antibiotics. We adapted E. faecalis OG1RF (a laboratory strain) and S613TM (a clinical strain) lacking liaR to increasing concentrations of DAP, leading to the development of DAP‐R and elevated MICs to bacitracin and ceftriaxone. Whole genome sequencing identified changes in the YxdJK two‐component regulatory system and a putative fatty acid kinase (dak) in both DAP‐R strains. Deletion of the gene encoding the YxdJ response regulator in both the DAP‐R mutant and wild‐type OG1RF decreased MICs to DAP, even when a functional LiaFSR system was present. Mutations in dak were associated with slower growth, decreased membrane fluidity and alterations of cell morphology. These findings suggest that overlapping stress response pathways can provide protection against antimicrobial peptides in E. faecalis at a significant cost in bacterial fitness.     

Characterization of an ethanol‐tolerant 1,4‐β‐xylosidase produced by Pichia membranifaciens

Aims: The purification and biochemical properties of the 1,4‐β‐xylosidase of an oenological yeast were investigated. Methods and Results: An ethanol‐tolerant 1,4‐β‐xylosidase was purified from cultures of a strain of Pichia membranifaciens grown on xylan at 28°C. The enzyme was purified by sequential chromatography on DEAE cellulose and Sephadex G‐100. The relative molecular mass of the enzyme was determined to be 50 kDa by SDS‐PAGE. The activity of 1,4‐β‐xylosidase was optimum at pH 6·0 and at 35°C. The activity had a K_m of 0·48 ± 0·06 mmol l^?1 and a V_max of 7·4 ± 0·1 μmol min^?1 mg^?1 protein for p‐nitrophenyl‐β‐d ‐xylopyranoside. Conclusions: The enzyme characteristics (pH and thermal stability, low inhibition rate by glucose and ethanol tolerance) make this enzyme a good candidate to be used in enzymatic production of xylose and improvement of hemicellulose saccharification for production of bioethanol. Significance and Impact of the Study: This study may be useful for assessing the ability of the 1,4‐β‐xylosidase from P. membranifaciens to be used in the bioethanol production process.     

Cloning of β-1,3-1,4-glucanase gene from Bacillus licheniformis EGW039 (CGMCC 0635) and its expression in Escherichia coli BL21 (DE3)

β-1,3-1,4-Glucanase has been applied in the brewing and animal feed additive industry. It can effectively improve digestibility of barley-based diets and reduce enteritis. It also reduces viscosity during mashing for high-quality brewers malt. The aim of this work is to clone β-1,3-1,4-glucanase-encoding gene and express it heterogeneously. The gene was amplified by polymerase chain reaction using Bacillus licheniformis genomic DNA as the template and ligated into the expression vector pET28a. The recombinant vector was transformed into Escherichia coli. The estimated molecular weight of the recombinant enzyme with a six-His tag at the N terminus was about 28 kDa, and its activities in cell lysate supernatant were 1,286 and 986 U ml⁻¹ for 1% (w/v) barley β-glucan and 1% (w/v) lichenan, respectively. Accordingly, the specific activities were 2,479 and 1,906 U mg⁻¹ for these two substrates. The expression level of recombinant β-1,3-1,4-glucanase was about 60.9% of the total protein and about 12.5% of the total soluble protein in crude cell lysate supernatant. Acidity and temperature optimal for this recombinant enzyme was pH 5.6 and 40°C, respectively.     

Production of N-Succinyl-l-diaminopimelic Acid by Auxotrophic Mutants of Aerobacter aerogenes and Serratia marcescens

α,ε-Diaminopimelic acid (DAP)-requiring mutants isolated from Aerobacter aerogenes ATCC 8308 and Serratia marcescens ATCC 19180 were found to accumulate N-succinyl-l-diaminopimelic acid (SDAP) which was an intermediate in the biosynthesis of lysine in Escherichia coli. SDAP was isolated from the culture broth and identified by the behavior in paper chromatography, melting point, elementary analysis, infrared spectrum, and optical rotation.

The culture conditions for SDAP production by A. aerogenes KY 7049 (DAP^?) and S. marcescens KY 8921 (DAP^?/Lys^?) were investigated. A. aerogenes KY 7049 has an absolute requirement for DAP together with a relative requirement for l-lysine. High levels of DAP (2000~4000 μg/ml) were proved to be favorable for SDAP accumulation, while if lysine along with DAP was added to the fermentation medium, optimal level of DAP for SDAP production was relatively low (about 200 μg/ml at 200 μg/ml of lysine). A variety of compounds which may conceivably affect the course of a fermentation process, i.e., carbon source, inorganic nitrogen source, amino acids, vitamines, precursors, were screened at optimal levels of lysine and DAP. Thus, the amount of SDAP accumulation reached a level of 19.9 mg/ml with the medium containing 10% glucose and 2000 μg/ml of DAP. S. marcescens KY 8921 requires either DAP or lysine for growth. Optimal level of DAP and lysine for SDAP accumulation was 50~100μg/ml.     

The timing of diammonium phosphate supplementation of wine must affects subsequent H2S release during fermentation

Aims:  The aim of this study was to evaluate the impact of supplementation by diammonium phosphate (DAP) on hydrogen sulfide (H₂S) production, when DAP given either prior to fermentation or during the early stationary growth phase of yeast. Methods and Results:  Three contrasting Saccharomyces cerevisiae wine strains were used to ferment synthetic grape juice (GJ) containing 67 mg l^?1 of initial yeast assimilable nitrogen (YAN), supplied either as DAP or as mixture of amino acids. Sufficient DAP was added either prior to or 72 h after the initiation of fermentation to achieve a final YAN concentration of 267 mg l^?1. Supplementation prior to fermentation stimulated H₂S production. The results obtained in model solutions were validated using natural GJ. Conclusion:  The timing of DAP supplementation is critical for ensuring that fermentation proceeds without excessive release of H₂S. Significance and Impact of the Study:  This result has important implications for the wine‐making industry, because it highlights the value of determining the initial nitrogen level of a GJ. It raises awareness of the dependence of wine quality on the correct timing of DAP supplementation.     

Co-inoculation of Urea and DAP Tolerant <Emphasis Type="Italic">Sinorhizobium meliloti</Emphasis> and <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> as Integrated Approach for Growth Enhancement of <Emphasis Type="Italic">Brassica juncea</Emphasis>

Two plant growth promoting rhizobacteria––Sinorhizobium meliloti RMP1 and Pseudomonas aeruginosa GRC₂ were studied for integrated nutrient management to obtain improved yield of Brassica juncea. Low concentrations of urea and diammonium phosphate (DAP) stimulated the growth of both S. meliloti RMP1 and P. aeruginosa GRC₂. 1 M of urea and 0.35 M of DAP was found lethal for RMP1, while 1.3 M and 0.37 M concentrations of urea and DAP proved to be toxic for GRC₂. Lc₅₀ was observed as 0.49 M of urea and 0.15 M of DAP for RMP1, and 0.66 M urea and 0.18 M of DAP for GRC₂. Urea and DAP adaptive variants of RMP1 and GRC₂ was isolated. Adaptive bacterial variants had better growth rates at sub-lethal (Lc₅₀) concentrations of urea and DAP as compared to non-adaptive variants. They also retained plant growth promoting attributes similar to non adaptive variants. GRC₂ and RMP1 did not affect the growth of each other and were chemotactically active for DAP, urea as well as root exudates of B. juncea. Both the isolates colonized well in the rhizosphere of B. juncea, as their populations were recorded ≈5 log₁₀ cfu g⁻¹ after 120 days. Interestingly, the colonization ability was found even better when both strains were co-inoculated, as their population was recorded in the range of ≈6 log₁₀ cfu g⁻¹ after 120 days. In field trials, application of RMP1 and GRC₂ resulted in significant increase in biomass and yield of B. juncea as compared to control. However, yield was better with application of half dose and full dose of recommended fertilizers. Interestingly, the biomass as well as yield improved further when both isolates were applied together along with half dose of recommended fertilizers.     

High-level expression of a specific β-1,3-1,4-glucanase from the thermophilic fungus Paecilomyces thermophila in Pichia pastoris

In this study, a novel β-1,3-1,4-glucanase gene (designated as PtLic16A) from Paecilomyces thermophila was cloned and sequenced. PtLic16A has an open reading frame of 945 bp, encoding 314 amino acids. The deduced amino acid sequence shares the highest identity (61%) with the putative endo-1,3(4)-β-glucanase from Neosartorya fischeri NRRL 181. PtLic16A was cloned into a vector pPIC9K and was expressed successfully in Pichia pastoris as active extracellular β-1,3-1,4-glucanase. The recombinant β-1,3-1,4-glucanase (PtLic16A) was secreted predominantly into the medium which comprised up to 85% of the total extracellular proteins and reached a protein concentration of 9.1 g l⁻¹ with an activity of 55,300 U ml⁻¹ in 5-l fermentor culture. The enzyme was then purified using two steps, ion exchange chromatography, and gel filtration chromatography. The purified enzyme had a molecular mass of 38.5 kDa on SDS–PAGE. It was optimally active at pH 7.0 and a temperature of 70°C. Furthermore, the enzyme exhibited strict specificity for β-1,3-1,4-d-glucans. This is the first report on the cloning and expression of a β-1,3-1,4-glucanase gene from Paecilomyces sp.     

Food consumption of Sarotherodon mossambicus (Trewaves) exposed to sublethal concentration of diammonium phosphate

S. mossambicus was exposed to toxic and sublethal concentrations of the fertilizer diammonium phosphate (0.2 to 1.0 g l^–1). Mortality, food utilization and growth were studied. At a concentration of 0.6 g l^–1 DAP, 100% mortality was observed within 96 h; no mortality occurred at 0.5 g l^–1; LC50 was 0.55 g l^–1. Rearing the fish in increasing sublethal concentrations of DAP, it was found that the feeding rate decreased from 25.4 mg g^–1 fish^–1 d^–1 (fish reared in DAP-free water) to 10.1 mg g^–1 d^–1 at the highest sublethal concentration (0.5 g l^–1). Growth rate was drastically reduced. At high sublethal concentrations of DAP, the fish lost reserve energy, in addition to the energy obtained from food intake for survival, as a result of increased swimming activity and opercular beats.     

Dipeptidyl Aminopeptidase IV from Pseudomonas sp. WO24

Dipeptidyl aminopeptidase IV from Pseudomonas sp. WO24 was purified as two molecular forms of 84 and 82-kDa by SDS–PAGE. Peptide mapping and N-terminal sequence analyses indicated that both proteins might be derived from the same protein, and that the 82-kDa molecule might be a truncated form from the 84-kDa molecule at least at the N-terminus. The DAP IV gene of Pseudomonas sp. WO24 was cloned and expressed in E. coli. The enzyme expressed in E. coli JM109 harboring a hybrid plasmid, pYO-6A, with about a 3-kbp fragment containing the DAP IV gene, was purified with an activity recovery of 24%. The recombinant enzyme also had the same two molecular forms, though the ratio of the two forms (about 1:1) was different from that of the native ones (about 1:4). The native and recombinant enzyme preparations had similar specific activities, suggesting that the 84 and 82-kDa molecules are in an active form and have almost the same specific activity. The molecular mass, the subunit number, the substrate specificity, and the effects of various inhibitors of the native enzyme indicated that this enzyme was a typical DAP IV and had properties similar to those of Flavobacterium meningosepticum rather than others.     

Seed development and vivipary in Zea mays L.

Seed development was investigated in kernels of developing wild-type and viviparous (vp-1) Zea mays L. Embryos and endosperm of wild-type kernels began to dehydrate at approx. 35 d after pollination (DAP); viviparous embryos did not desiccate but accumulated fresh weight via coleoptile growth in the caryopses. Concentrations of endogenous abscisic acid (ABA) in the embryo were relatively high early in development, being approx. 150 ng·g^-1 fresh weight at 20 DAP. The ABA content declined thereafter, falling to approx. 50 ng·g^-1 at 30 DAP. Endosperm ABA content was always low, being less than 20 ng·g^-1. There were no differences between wild-type and vp-1 tissues. Immature kernels did not germinate when removed from the ear until late in development. The ability to germinate was correlated with decreasing moisture content in the endosperm at the time of removal; premature drying of immature kernels resulted in greatly increased germination following imbibition. Excised embryos germinated precociously when removed from the endosperm as early as 25 DAP. Such germination could be prevented by treatment with 10^-5 M ABA or by lowering the solute potential (_s) of the medium with 0.3 M mannitol. Treatment of excised embryos with ABA led to internal ABA concentrations comparable to those in embryos in which germination was inhibited in situ. Mannitol treatment did not have this effect, although water-deficit stress of excised embryos resulted in substantial ABA production. Germinated vp-1 embryos were less sensitive to growth inhibition by ABA or mannitol than germinating wild-type embryos. The vp-1 seedlings were not wilty and their transpiration rates were reduced in response to ABA or water shortage.Abbreviations and symbols ABA abscisic acid - DAP days after pollination - FW fresh weight - vp-1 viviparous genotype - _s solute potential     

Leupeptins Produced by the Marine Alteromonas sp. B-10–31

Endo-1,4-β-D-mannanase (1,4-β-D-mannanohydrolase, EC 3.2.1.78) was purified from viscera of a mud snail, Pomacea insularus (de Ordigny). The purified enzyme gave a single protein band in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight of the purified enzyme was estimated to be 44,000. The amino-terminal sequence was H· Gly-X-Leu-Arg-Arg-Gln-Gly-Thr-Asn-Ile-Val-Asp-Ser-His-Gly-His-Lys-Val-Phe-Leu-Ser-Gly-Ala-Asn-Thr-Ala-Trp-Val-Ala-Tyr-Gly-Tyr-Asp-. The enzyme was stable from pH about 5.0 to about 10.5 and had its maximum activity at pH about 5.5. The purified enzyme produced M2, M3, M4,and M5 from β-1,4-mannan. Enzyme activity was greatly inhibited by Ag⁺, Hg²⁺, Cu²⁺, and dithiothreitol at 1 mM concentration. In addition, N-bromosuccinimide completely inhibited the enzyme activity.     

Nitrogen fixation and growth of soybean as influenced by varying the methods of inoculation with Bradyrhizobium japonicum

The effects of inoculating soil with a water suspension of Bradyrhizobium japonicum (i) at seeding, (ii) 7, or (iii) 14 days after planting (DAP), (iv) seed slurry inoculation and (v) seed slurry supplemented with postemergence inoculation of a water suspension of Bradyrhizobium at 7 or (vi) 14 DAP, on nodulation, N₂ fixation and yield of soybean (Glycine max. [L.] Merrill) were compared in the greenhouse. The ¹⁵N isotope dilution technique was used to quantify N₂ fixed at flowering, early pod filling and physiological maturity stages (36, 52 and 70 DAP, respectively). On average, the water suspension inoculation formed the greatest number of nodules, and seed plus postemergence inoculation formed slightly more nodules than the seed-only inoculated plants (27, 19 and 12 nodules/plant respectively at physiological maturity). Seed slurry inoculation followed by postemergence inoculation at 14 DAP gave the highest nodule weight, with the plants fixing significantly more (P<0.05) N₂ (125 mg N plant⁻¹ or 56% N) than any other treatment (mean, 75 mg plant⁻¹ or 35% N). However, the higher N₂ fixation was not translated into higher N or dry matter yields. Estimates of N₂ fixed by the ostemergence Bradyrhizobium inoculations as well as plant yield were not significantly different from those of the seed slurry inoculation. Thus, delaying inoculation (e.g., by two weeks as in this study) did not reduce the symbiotic ability of soybean plants.