The ammonotelic African lungfish,<Emphasis Type="Italic"> Protopterus dolloi</Emphasis>, increases the rate of urea synthesis and becomes ureotelic after feeding

This study aimed to elucidate the role of urea synthesis in the slender African lungfish Protopterus dolloi in detoxifying ammonia after feeding. There were significant increases in the rate of ammonia excretion in P. dolloi between hours 6 and 15 after feeding. Simultaneously, there were significant increases in urea excretion rates between hours 3 and 18. Consequently, the percentage of total nitrogen (N) excreted as urea N increased to ~60% between hours 12 and 21 post-feeding. Hence, after feeding, the normally ammonotelic P. dolloi became ureotelic. Approximately 41% of the N intake from food was excreted within 24 h by P. dolloi, 55% of which was in the form of urea N. At hour 12 post-feeding, the accumulation of urea N was greater than the accumulation of ammonia N in various tissues, which indicates that feeding led to an increase in the rate of urea synthesis. This is contrary to results reported previously on the infusion of ammonia into the peritoneal cavity of the marine dogfish shark, in which a significant portion of the exogenous ammonia was excreted as ammonia. In contrast, feeding is more likely to induce urea synthesis, which is energy intensive, because feeding provides an ample supply of energy resources and leads to the production of ammonia intracellularly in the liver. The capacity of P. dolloi to synthesize urea effectively prevented a postprandial surge in the plasma ammonia level as reported elsewhere for other non-ureogenic teleosts. However, there was a significant increase in the glutamine content in the brain at hour 24, indicating that the brain had to defend against ammonia toxicity after feeding.Communicated by I.D. Hume     

Increased urea synthesis and/or suppressed ammonia production in the African lungfish, Protopterus annectens, during aestivation in air or mud

The objective of this study was to elucidate how the African lungfish, Protopterus annectens, ameliorated ammonia toxicity during 12 or 46 days of aestivation in air or in mud. Twelve days of aestivation in air led to significant increases in contents of urea, but not ammonia, in tissues of P. annectens. The estimated rate of urea synthesis increased 2.7-fold despite the lack of changes in the activities of hepatic ornithine–urea cycle enzymes, but there was only a minor change in the estimated rate of ammonia production. After 46 days of aestivation in air, the ammonia content in the liver decreased significantly and contents of urea in all tissues studied increased significantly, indicating that the fish shifted to a combination of increased urea synthesis (1.4-fold of the day 0 value) and decreased ammonia production (56% of the day 0 value) to defend against ammonia toxicity. By contrast, 12 days of aestivation in mud produced only minor increases in tissue urea contents, with ammonia contents remained unchanged. This was apparently achieved through decreases in urea synthesis and ammonia production (40 and 15%, respectively, of the corresponding day 0 value). Surprisingly, 46 days of aestivation in mud resulted in no changes in tissue urea contents, indicating that profound suppressions of urea synthesis and ammonia production (2.6 and 1.2%, respectively, of the corresponding day 0 value) had occurred. This is the first report on such a phenomenon, and the reduction in ammonia production was so profound that it could be the greatest reduction known among animals. Since fish aestivated in mud had relatively low blood pO₂ and muscle ATP content, they could have been exposed to hypoxia, which induced reductions in metabolic rate and ammonia production. Consequently, fish aestivating in mud had a lower dependency on increased urea synthesis to detoxify ammonia, which is energy intensive, than fish aestivating in air.     

The effect of temperature on post-feeding ammonia excretion and oxygen consumption in the southern catfish

The post-prandial rates of ammonia excretion (TAN) and oxygen consumption in the southern catfish (Silurus meridionalis) were assessed at 2 h intervals post-feeding until the rates returned to those of the fasting rates, at 17.5, 22.5, 27.5, and 32.5°C, respectively. Both fasting TAN and increased with temperature, and were lower than those previously reported for many fish species. The relationship between fasting TAN (mmol NH₃–N kg⁻¹ h⁻¹) and temperature (T, °C) was described as: fasting TAN = 0.144e ^0.0266T (r = 0.526, n = 27, P < 0.05). The magnitude of ammonia excretion and its ratio to total N intake during the specific dynamic action (SDA) tended to increase initially, and then decrease with increasing temperature. The ammonia quotient (AQ), calculated as mol NH₃–N/mol O₂, following feeding decreased as temperature increased. The relationship between AQ during SDA and temperature was described as: AQ_{during SDA} = 0.303e ^−0.0143T (r = 0.739, n = 21, P < 0.05). Our results suggest that ammonia excretion and oxygen consumption post-feeding are operating independently of each other. Furthermore, it appears that the importance of protein as a metabolic substrate in postprandial fish decreases with temperature.     

Production of saponions and polysaccharide in the presence of lactoalbumin hydrolysate in <Emphasis Type="Italic">Panax quinquefolium</Emphasis> L. cells cultures

In this article, ginsenosides and polysaccharide contents in suspension cells and native roots of Panax quinquefolium L. were studied. In order to enhance the contents of ginsenosides and polysaccharide in P. quinquefolium suspension cells, we tested the effects of lactoalbumin hydrolysate on the growth of P. quinquefolium suspension cell, synthesis of ginsenosides and polysaccharide in flask and bioreactor. In flask culture, cells growth ratio was significantly enhanced by the addition of lower concentration of lactoalbumin hydrolysate. Addition of 100 mg L⁻¹ lactoalbumin hydrolysate significantly enhanced the contents of total saponins (5.44 mg g⁻¹ DW) and the contents were 3.89-fold over the control group. Addition of lactoalbumin hydrolysate significantly promoted the accumulation of polysaccharide, except 200 mg L⁻¹ lactoalbumin hydrolysate. The highest total saponins yield (36.72 mg L⁻¹ DW) and polysaccharide yield (0.83 g L⁻¹ DW) were obtained at 100 mg L⁻¹ lactoalbumin hydrolysate. In a 5-L stirred tank bioreactor, the highest contents of total saponins and TRb group ginsenosides were achieved on day 26, while the effect of lactoalbumin hydrolysate on the contents of TRg group ginsenosides were insignificant. This result suggests that lactoalbumin hydrolysate might have triggered the enzyme activities for the synthesis of TRb group ginsenosides. Overall, the highest total saponins yield (31.37 mg L⁻¹ DW) and polysaccharide yield (1.618 g L⁻¹ DW) were obtained on day 26 and day 24 respectively and the polysaccharide yield was 1.95-fold higher than the shake flask culture (0.83 g L⁻¹ DW). These results provided theoretical reference for two-stage culture in suspension cells of P. quinquefolium in bioreactor.     

Analysis of two promoters that control the expression of the <Emphasis Type="Italic">GTP cyclohydrolase I</Emphasis> gene in <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

GTP cyclohydrolase I (GTPCH) is a key enzyme in the de novo synthesis of tetrahydrobiopterin. Previously, the Drosophila melanogaster GTPCH gene has been shown to be expressed from two different promoters (P1 and P2). In our study, the 5′-flanking DNA regions required for P1 and P2 promoter activities were characterized using transient expression assay. The DNA regions between −98 and +31, and between −73 and +35 are required for efficient P1 and P2 promoter activities, respectively. The regions between −98 and −56 and between −73 and −41 may contain critical elements required for the expression of GTPCH in Drosophila. By aligning the nucleotide sequences in the P1 and P2 promoter regions of the Drosophila melanogaster and Drosophila virilis GTPCH genes, several conserved elements including palindromic sequences in the regions critical for P1 and P2 promoter activities were identified. Western blot analysis of transgenic flies transformed using P1 or P2 promoter-lacZ fusion plasmids further revealed that P1 promoter expression is restricted to the late pupae and adult developmental stages but that the P2 promoter driven expression of GTPCH is constitutive throughout fly development. In addition, X-gal staining of the embryos and imaginal discs of transgenic flies suggests that the P2 promoter is active from stage 13 of embryo and is generally active in most regions of the imaginal discs at the larval stages.     

Potential of active excretion of ammonia in three different haline species of crabs

Isolated perfused gills of stenohaline crabs Cancer pagurus adapted to seawater, brackish water-adapted euryhaline shore crabs Carcinus maenas and freshwater-adapted extremely euryhaline Chinese crabs Eriocheir sinensis were tested for their capacity to excrete ammonia. Gills were perfused with haemolymph-like salines and bathed with salines equal in adaptation osmolality. Applying 100 μmol · l⁻¹ NH₄Cl in the perfusion saline and concentrations of NH₄Cl in the bath that were stepwise increased from 0 to 4000 μmol · l⁻¹ allowed us to measure transbranchial fluxes of ammonia along an outwardly as well as various inwardly directed gradients. The gills of all three crab species were capable – to different extents – of active excretion of ammonia against an inwardly directed gradient. Of the three crab species, the gills of Cancer pagurus revealed the highest capacity for active excretion of ammonia, being able to excrete it from the haemolymph (100 μmol · l⁻¹ NH⁺ ₄) through the gill epithelium against ambient concentrations of up to 800 μmol · l⁻¹, i.e. against an eightfold gradient. Carcinus maenas and E. sinensis were able to actively excrete ammonia against approximately fourfold gradients. Within the three crab species, the gills of E. sinensis exhibited the greatest capacity to resist influx at very high external concentrations of up to 4000 μmol · l⁻¹. We consider the observed capacities for excretion of ammonia against the gradient as ecologically meaningful. These benthic crustaceans protect themselves by burying themselves in the sediment, where, in contrast to the water column, concentrations of ammonia have previously been reported that greatly increase haemolymph levels. Electrophysiological results indicate that the permeabilities of the gill epithelia are a clue to understanding the species-specific differences in active excretion of ammonia. During the invasion of brackish water and freshwater, the permeabilities of the body surfaces greatly decreased. The gills of marine Cancer pagurus exibited the greatest permeability (ca. 250 mS cm⁻²), thus representing practically no influx barrier for ions including NH⁺ ₄. We therefore assume that C. pagurus had to develop the strongest mechanism of active excretion of ammonia to counteract influx. On the other hand, freshwater-adapted E. sinensis exhibited the lowest ion permeability (ca. 4 mS cm⁻²) which may reduce passive NH⁺ ₄ influxes at high ambient levels. Accepted: 14 October 1998     

Identification and characterization of a novel nitrilase from <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> Pf-5

Nitrile groups are catabolized to the corresponding acid and ammonia through one-step reaction involving a nitrilase. Here, we report the use of bioinformatic and biochemical tools to identify and characterize the nitrilase (NitPf5) from Pseudomonas fluorescens Pf-5. The nitPf5 gene was identified via sequence analysis of the whole genome of P. fluorescens Pf-5 and subsequently cloned and overexpressed in Escherichia coli. DNA sequence analysis revealed an open-reading frame of 921 bp, capable of encoding a polypeptide of 307 amino acids residues with a calculated isoelectric point of pH 5.4. The enzyme had an optimal pH and temperature of 7.0°C and 45°C, respectively, with a specific activity of 1.7 and 1.9 μmol min⁻¹ mg protein⁻¹ for succinonitrile and fumaronitrile, respectively. The molecular weight of the nitrilase as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration chromatography was 33,000 and 138,000 Da, respectively, suggesting that the enzyme is homotetrameric. Among various nitriles, dinitriles were the preferred substrate of NitPf5 with a K _m = 17.9 mM and k _cat/K _m = 0.5 mM⁻¹ s⁻¹ for succinonitrile. Homology modeling and docking studies of dinitrile and mononitrile substrate into the active site of NitPf5 shed light on the substrate specificity of NitPf5. Although nitrilases have been characterized from several other sources, P. fluorescens Pf-5 nitrilase NitPf5 is distinguished from other nitrilases by its high specific activity toward dinitriles, which make P. fluorescens NitPf5 useful for industrial applications, including enzymatic synthesis of various cyanocarboxylic acids.     

Effects of temperature and ammonium on growth,pigment production and nitrogen uptake by four species of <Emphasis Type="BoldItalic">Porphyra</Emphasis> (Bangiales,Rhodophyta) native to the New England coast

Porphyra is one of the world’s most valued maricultured seaweeds and has been cultivated for several hundred years in Asia. The objective of this study was to produce critical information as a guide for the selection of an appropriate Porphyra species from coastal New England for the development of a land-based aquaculture system. Four Northwest Atlantic Porphyra species: P. leucosticta, P. amplissima, P. linearis and P. umbilicalis, were cultivated for 1 and 2 weeks at saturated light intensities (100–150 μmol photons m⁻²s⁻¹) and six combinations of ammonium (25 and 250 μmoles L⁻¹) and temperature (10, 15 and 20°C). Specific growth rate (SGR) increased with decreasing temperature in P. leucosticta, P. linearis and P. umbilicalis and increased with increasing temperature in P. amplissima. The SGR of all species was greater at the higher ammonium concentration. Porphyra linearis had the highest SGR, increasing in biomass by approximately 16% day⁻¹. Phycoerythrin (PE) content was higher at 10°C and 250 μmoles L⁻¹ in all species except P. amplissima. The PE content, measured as fresh weight (FW), of P. linearis (29 mg g⁻¹ FW⁻¹) and P. umbilicalis (26 mg g⁻¹ FW⁻¹) was significantly higher than the other two species. Tissue nitrogen content of all species measured in dry weight was on average 1.45% higher at 250 μmoles L⁻¹ than at 25 μmoles L⁻¹ ammonium concentration. Porphyra umbilicalis had the highest tissue nitrogen contents (6.76%) at 10°C and 250 μmoles L⁻¹ ammonium. Based on these results, P. linearis and P. umbilicalis should be considered as potential candidates for bioremediation with finfish and shellfish mariculture.     

Enhancement of polysaccharides production in <Emphasis Type="Italic">Ganoderma lucidum</Emphasis> by the addition of ethyl acetate extracts from <Emphasis Type="Italic">Eupolyphaga sinensis</Emphasis> and <Emphasis Type="Italic">Catharsius molossus</Emphasis>

To screen stimulators from Chinese medicinal insects for mycelial growth and polysaccharides production of Ganoderma lucidum, G. lucidum was inoculated into the media with and without supplementation of medicinal insect extracts. The ethyl acetate extract of Eupolyphaga sinensis at 55 mg l⁻¹ lead to significant increase in both biomass and intracellular polysaccharides (IPS) concentration from 8.53 ± 0.41 to 14.16 ± 0.43 and 1.28 ± 0.09 to 2.13 ± 0.11 g l⁻¹, respectively. In addition, the ethyl acetate extract of Catharsius molossus at 55 mg l⁻¹ significantly enhanced extracellular polysaccharides (EPS) production; the EPS yield increased from 350.9 ± 14.1 to 475.1 ± 15.3 mg l⁻¹. There were no new components in the two types of polysaccharides obtained by the addition of the insect extracts.     

Effects of intra-peritoneal injection with NH4Cl, urea, or NH4Cl+urea on nitrogen excretion and metabolism in the African lungfish Protopterus dolloi

This study aimed to (1) determine if ammonia (as NH(4)Cl) injected intra-peritoneally into the ureogenic slender African lungfish, Protopterus dolloi, was excreted directly rather than being converted to urea; (2) examine if injected urea was retained in this lungfish, leading to decreases in liver arginine and brain tryptophan levels, as observed during aestivation on land; and (3) elucidate if increase in internal ammonia level would affect urea excretion, when ammonia and urea are injected simultaneously into the fish. Despite being ureogenic, P. dolloi rapidly excreted the excess ammonia as ammonia within the subsequent 12 h after NH(4)Cl was injected into its peritoneal cavity. Injected ammonia was not detoxified into urea through the ornithine-urea cycle, probably because it is energetically intensive to synthesize urea and because food was withheld before and during the experiment. In addition, injected ammonia was likely to stay in extracellular compartments available for direct excretion. At hour 24, only a small amount of ammonia accumulated in the muscle of these fish. In contrast, when urea was injected intra-peritoneally into P. dolloi, only a small percentage (34%) of it was excreted during the subsequent 24-h period. A significant increase in the rate of urea excretion was observed only after 16 h. At hour 24, significant quantities of urea were retained in various tissues of P. dolloi. Injection with urea led to an apparent reduction in endogenous ammonia production, a significant decrease in the hepatic arginine content, and a significantly lower level of brain tryptophan in this lungfish. All three phenomena had been observed previously in aestivating P. dolloi. Hence, it is logical to deduce that urea synthesis and accumulation could be one of the essential factors in initiating and perpetuating aestivation in this lungfish. Through the injection of NH(4)Cl + urea, it was demonstrated that an increase in urea excretion occurred in P. dolloi within the first 12 h post-injection, which was much earlier than that of fish injected with urea alone. These results suggest that urea excretion in P. dolloi is likely to be regulated by the level of internal ammonia in its body.     

Reinvasion of a Riparian Forest Community by an Animal-dispersed Tree Weed Following Control Measures

Patterns of seed dispersal and the effects of mulching upon Celtis sinensis Pers. seedling establishment were investigated following the removal of this tree weed from a riparian forest community. At the commencement of the study there was virtually no representation of C. sinensis in the soil seed bank. However, subsequent rates of seed immigration were high since mature individuals of C. sinensis remained on the boundary of the site. Fruit bats (Pteropus spp.) were the principal dispersal vectors. Seed rain density of C. sinensis was best fitted by an inverse power distribution, with seed densities in excess of 20 m⁻² detected at 70 m from the seed source. Extrapolation from this relationship suggested that a site would have to be more than 350 m from a seed source to reduce the C. sinensis seed rain to less than 1 m⁻². More than 98% of the seed rain occurred below the canopies of the native tree species that remained following the removal of C. sinensis. For these trees, subarboreal C. sinensis seed distributions were not homogeneous, with peak seed densities occurring at different distances from tree trunks in each of the two years that seed distributions were assessed. Mulching with compacted sugar cane trash, corresponding to litter loadings of 6–12 kg m⁻², was imposed early in the study, some weeks before the C. sinensis seed rain commenced. These treatments had no measurable effect upon C. sinensis germination, but substantially reduced seedling survival and had variable effects upon the early growth of seedlings. The potential roles of seed limitation vs establishment limitation are discussed in relation to the management of animal-dispersed invasive species. It is argued that an understanding of the likely levels and patterns of invasion is essential for the formulation of management strategies that can effectively reduce the invasion and impacts of these plants. This revised version was published online in July 2006 with corrections to the Cover Date.     

Astaxanthin production by <Emphasis Type="Italic">Phaffia rhodozyma</Emphasis> and <Emphasis Type="Italic">Haematococcus pluvialis</Emphasis>: a comparative study

Phaffia rhodozyma (now Xanthophyllomyces dendrorhous) and Haematococcus pluvialis are known as the major prominent microorganisms able to synthesize astaxanthin natural pigment. Important research efforts have been made to determine optimal conditions for astaxanthin synthesis. When the focus is on astaxanthin production, the maximal reported value of 9.2 mg/g cell is obtained within H. pluvialis grown on BAR medium, under continuous illumination (345 μmol photon m⁻² s⁻¹) and without aeration. Whereas fermentation by mutated R1 yeast grown on coconut milk produced 1,850 μg/g yeast. However, when looking at astaxanthin productivity, the picture is slightly different. The figures obtained with P. rhodozyma are rather similar to those of H. pluvialis. Maximal reported values are 170 μg/g yeast per day with a wild yeast strain and 370 μg/g yeast per day with mutated R1 yeast. In the case of H. pluvialis, maximal values ranged from 290 to 428 μg/g cell per day depending on the media (BG-11 or BAR), light intensity (177 μmol photon m⁻² s⁻¹), aeration, etc. The main aim of this work was to examine how astaxanthin synthesis, by P. rhodozyma and H. pluvialis, could be compared. The study is based on previous works by the authors where pigment productions have been reported.     

Changes in fatty acids and sterols during batch growth of <Emphasis Type="Italic">Pavlova viridis</Emphasis> in photobioreactor

Changes in the composition of fatty acids and sterols of Pavlova viridis cultured in an air-lift photobioreactor were studied using gas chromatography-mass spectrometry (GC-MS). The results show that radical changes in fatty acid and sterol contents and compositions occurred during growth phase transitions: the total lipid increased along with the culture age, from 166.4 mg g⁻¹ (late exponential phase) to 232.7 mg g⁻¹ (linear phase), and increased further to be 235.1 mg g⁻¹ in the stationary phase. Polyunsaturated fatty acids (PUFAs), especially eicosapentaenoic acid (EPA), decreased along with the culture time, PUFAs, and EPA contents maximized in the late exponential phase to become 46.2 mg g⁻¹ and 22.1 mg g⁻¹ respectively; there was no significant change in docosahexaenoic acid (DHA) content during the whole growth phase, although it reached the peak in the linear phase with 3.5 mg g⁻¹. As for the sterols, two unique sterols with two hydroxyl groups, termed pavlovols, were observed. 4α,24-Dimethylcholestan-3β,4β-diol, one of the pavlovols, increased almost 2-fold from the late exponential phase (2.5 mg g⁻¹) to the stationary phase (4.3 mg g⁻¹). On the contrary, the contents of stigmasterol and sitosterol decreased with culture age, with the maximum content of 2.4 mg g⁻¹ and 3.1 mg g⁻¹, both obtained in the late exponential phase, respectively. The results indicate that growth phase control could be used as a methodology to optimize the total lipid, EPA, PUFA, and sterol contents with the potential for both aquaculture feeds and nutraceutical applications, especially for further research into unique pavlovols.     

Lipopolysaccharide of Escherichia coli, polyamines, and acetic acid stimulate cell proliferation in intestinal epithelial cells

Summary Our aim was to examine whether lipopolysaccharide of Escherichia coli, polyamines of dietetic and/or bacterial origin, and products of the bacterial metabolism influence cell proliferation in epithelial cells from the colon and small intestine. Lipopolysaccharide of Escherichia coli 0111:B4 was incubated with cultures from human colonic mucosa. The mitoses were arrested with Vincristine and the total number of metaphases per crypt was counted. In addition, lipopolysaccharide was incubated with a human colonic epithelial cell line from adenocarcinoma (LS-123 cells) and with a nontransformed small intestinal cell line from germ-free rats (IEC-6 cells) for 24 h. In the last 4 h, the cells were labeled with tritiated thymidine. The cells were incubated with putrescine, cadaverine, and spermidine at 10⁻¹¹–10⁻³ M and with acetic acid (10⁻⁵–10⁻¹ M), acetaldehyde (10⁻¹⁰–10⁻⁴ M) and ammonium chloride (1–20 mM). Lipopolysaccharide of Escherichia coli increased the number of arrested metaphases in human colonic crypts and DNA synthesis in L-123 and IEC-6 cells (P<0.001). All polyamines increased DNA synthesis in the colonic and small intestinal cell lines, the effects being more marked for putrescine (P<0.001). The higher concentrations of acetic acid increased DNA synthesis in both epithelial cell lines (P<0.001). Acetaldehyde slightly decreased DNA synthesis in LS-123 cells at cytotoxic concentrations. Ammonium chloride did not significantly affect DNA synthesis. The final concentration of nonionized ammonia was less than 3%. It is concluded that lipopolysaccharides of Escherichia coli and intraluminal factors derived from microorganisms increase cell proliferation in human colonic crypts and intestinal epithelial cell lines.     

The effect of substituted amides of pyrazine-2-carboxylic acids on flavonolignan production in <Emphasis Type="Italic">Silybum marianum</Emphasis> culture <Emphasis Type="Italic">in vitro</Emphasis>

In vitro plant tissue and cell cultures were used to study herbicide effects on growth, selected metabolic activities and other phenomena. The effect of abiotic elicitors, two newly synthesized substituted amides of pyrazine-2-carboxylic acids (synthesized at the Department of Pharmaceutical Chemistry and Drug Control, School of Pharmacy in Hradec Kralove), on the flavonolignan accumulation in callus and suspension culture of Silybum marianum (L.) Gaertn. was investigated. The compounds markedly influenced production of flavonolignans in an in vitro culture. Particularly after the elicitation with a solution of compound 3-methylamide 5-tert-butylpyrazine-2-carboxylic acid at a concentration of 3.71×10⁻⁷ mol 1⁻¹ and within 72 h of elicitation, an increase in flavonolignan production by 893 % in suspension culture versus control took place. The flavonolignan accumulation in callus culture after the elicitation with a solution of 5-brom-2-hydroxyphenylamide of 5-tert-butyl-6-chloropyrazine-2-carboxylic acid was also increased by about 1039% (24 hour elicitation and concentration of 2.59×10⁻⁴ mol 1⁻¹).     

Effects of peritoneal injection of NH4HCO3 on nitrogen excretion and metabolism in the swamp eel Monopterus albus-- increased ammonia excretion with an induction of glutamine synthetase activity

Monopterus albus has to deal with high environmental ammonia concentrations during dry seasons and agricultural fertilization in rice fields. In this study, NH4HCO3 (10 micromol per g fish) was injected into the peritoneal cavity of M. albus, raising the level of ammonia in the body, in order to elucidate the strategies involved in defense against the toxicity of exogenous ammonia. During the subsequent 24 h after NH4HCO3 injection, there was a significant increase in the ammonia excretion rate, which indicates that the main strategy adopted by M. albus was to remove the majority of the exogenous ammonia through enhanced ammonia excretion. Exogenous ammonia was not detoxified into urea for excretion or accumulation. Six hours post-injection of NH4HCO3, ammonia content in the tissues built up significantly, especially in the brain, which suggests that M. albus had high tolerance of ammonia toxicity at the cellular and sub-cellular levels. By hour 12 post-injection, there were significant increases in the activities of glutamine synthetase in the muscle, liver, and gut, accompanied by significant increases in glutamine contents in the muscle and the liver. There was also a significant increase in the glutamine content in the brain at hour 6 post-injection of NH4HCO3. These results confirm the capability of M. albus to detoxify ammonia through glutamine synthesis. Overall, injection of NH4HCO3 had only minor effects on the contents of FAAs, other than glutamine, in tissues of M. albus because the majority (70%) of the injected ammonia was excreted within the 24-h period.     

Microbial production of <Emphasis Type="Italic">cis</Emphasis>-1,2-dihydroxy-cyclohexa-3,5-diene-1-carboxylate by genetically modified <Emphasis Type="Italic">Pseudomonas putida</Emphasis>

Arene cis-diols are interesting chemicals because of their chiral structures and great potentials in industrial synthesis of useful chiral chemical products. Pseudomonas putida KT2442 was genetically modified to transform benzoic acid (benzoate) to 1,2-dihydroxy-cyclohexa-3,5-diene-1-carboxylic acid (DHCDC) or named benzoate cis-diol. BenD gene encoding cis-diol dehydrogenase was deleted to generate a mutant named P. putida KTSY01. Genes benABC encoding benzoate dioxygenase were cloned into plasmid pSYM01 and overexpressed in P. putida KTSY01. The recombinant bacteria P. putida KTSY01 (pSYM01) showed strong ability to transform benzoate to DHCDC. DHCDC of 2.3 g/L was obtained with a yield of 73% after 24 h of cultivation in shake flasks incubated under optimized growth conditions. Transformation of benzoate carried out in a 6-L fermentor using a benzoate fed-batch process produced over 17 g/L DHCDC after 48 h of fermentation. The average DHCDC production rate was 0.356 g L⁻¹ h⁻¹. DHCDC purified from the fermentation broth showed a purity of more than 95%, and its chemical structure was confirmed by nuclear magnetic resonance.     

Drought,warming and soil fertilization effects on leaf volatile terpene concentrations in <Emphasis Type="Italic">Pinus halepensis</Emphasis> and <Emphasis Type="Italic">Quercus ilex</Emphasis>

The changes in foliar concentrations of volatile terpenes in response to water stress, fertilization and temperature were analyzed in Pinus halepensis and Quercus ilex. The most abundant terpenes found in both species were α-pinene and Δ³-carene. β-Pinene and myrcene were also abundant in both species. P. halepensis concentrations were much greater than those of Q. ilex in agreement with the lack of storage in the latter species (15205.60 ± 1140.04 vs. 0.54 ± 0.08 μg g⁻¹ [d.m.]). The drought treatment (reduction to 1/3 of full watering) significantly increased the total terpene concentrations in both species (54% in P. halepensis and 119% in Q. ilex). The fertilization treatment (addition of either 250 kg N ha⁻¹ or 250 kg P ha⁻¹ or both) had no significant effects on terpene foliar concentrations. The terpene concentrations increased from 0.25 μg g⁻¹ [d.m.] at 30°C to 0.70 μg g⁻¹ [d.m.] at 40°C in Q. ilex (the non-storing species) and from 2,240 μg g⁻¹ [d.m.] at 30°C to 15,621 μg g⁻¹ [d.m.] at 40°C in P. halepensis (the storing species). Both species presented negative relationship between terpene concentrations and relative water contents (RWC). The results of this study show that higher terpene concentrations can be expected in the warmer and drier conditions predicted for the next decades in the Mediterranean region.     

Improvement of growth,under field conditions,of wheat inoculated with <Emphasis Type="Italic">Pseudomonas chlororaphis</Emphasis> subsp.<Emphasis Type="Italic"> aurantiaca</Emphasis> SR1

Effects of inoculation of wheat (Triticum aestivum L.) with the rhizobacterium Pseudomonas chlororaphis subsp. aurantiaca strain SR1 (termed SR1) were studied at an experimental field site in Río Cuarto, Argentina. Treatments involved SR1 inoculation with or without nitrogen/phosphorus fertilization. Inoculation produced a significant increase in plant height and root length in early growth stages. Inoculation plus fertilization with 40 kg ha⁻¹ urea/30 kg ha⁻¹ diamonic phosphate (“50% dose”) gave a yield increase of 636 kg ha⁻¹ relative to control, and an increase of 472 kg ha⁻¹ relative to fertilization with 80 kg ha⁻¹ urea/60 kg ha⁻¹ phosphate without inoculation. SR1 inoculation without fertilization, compared to control, produced increases of 6% in weight of 1,000 grains, 13% in number of spikes per plant, and 30% in number of grains per spike. Inoculation plus 50% dose fertilization also improved these parameters. Results of the study indicate that inoculation of wheat with SR1 improves various growth and yield parameters, and allows reduced dosage of nitrogen/phosphorus fertilizers in the field.     

Comparative effectiveness of <Emphasis Type="Italic">Pseudomonas</Emphasis> and <Emphasis Type="Italic">Serratia</Emphasis> sp. containing ACC-deaminase for improving growth and yield of wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) under salt-stressed conditions

Ethylene synthesis is accelerated in response to various environmental stresses like salinity. Ten rhizobacterial strains isolated from wheat rhizosphere taken from different salt affected areas were screened for growth promotion of wheat under axenic conditions at 1, 5, 10 and 15 dS m⁻¹. Three strains, i.e., Pseudomonas putida (N21), Pseudomonas aeruginosa (N39) and Serratia proteamaculans (M35) showing promising performance under axenic conditions were selected for a pot trial at 1.63 (original), 5, 10 and 15 dS m⁻¹. Results showed that inoculation was effective even in the presence of higher salinity levels. P. putida was the most efficient strain compared to the other strains and significantly increased the plant height, root length, grain yield, 100-grain weight and straw yield up to 52, 60, 76, 19 and 67%, respectively, over uninoculated control at 15 dS m⁻¹. Similarly, chlorophyll content and K⁺/Na⁺ of leaves also increased by P. putida over control. It is highly likely that under salinity stress, 1-aminocyclopropane-1-carboxylic acid-deaminase activity of these microbial strains might have caused reduction in the synthesis of stress (salt)-induced inhibitory levels of ethylene. The results suggested that these strains could be employed for salinity tolerance in wheat; however, P. putida may have better prospects in stress alleviation/reduction.