'P' solubilization potential of plant growth promoting Pseudomonas mutants at low temperature

Pseudomonas fluorescens strain GRS₁, PRS₉ and their cold tolerant mutants were examined for their tricalcium phosphate (TCP) solubilizing activity in NBRIP (broth) media at 10°C and 25°C. Invariably, all the cold tolerant mutants of GRS₁ and PRS₉ were found more efficient than their respective wild type counterparts for ‘P’ solubilization activity at 10°C as compared to 25°C. ‘P’ solubilization potential of CRM was found maximum among all the strains followed by CRPF₆ and CRPF₄. To the best of out knowledge, this is the first report regarding low temperature ‘P’ solubilization activity.     

Air-breathing catfish, Clarias batrachus upregulates glutamine synthetase and carbamyl phosphate synthetase III during exposure to high external ammonia

We assessed the possible upregulation of glutamine synthetase (GS) and typical 'fish type' carbamyl phosphate synthetase III (CPS III) in detoxification of ammonia in different tissues of the walking catfish (Clarias batrachus) during exposure to 25 mM NH(4)Cl for 7 days. Exogenous ammonia led to an increase in ammonia and urea concentrations in different tissues. The results revealed the presence of relatively high levels of GS activity in the brain, liver and kidney, unexpectedly, also in the muscle, and even higher levels in the intestine and stomach. Exposure to high external ammonia (HEA) caused significant increase of activities of GS, CPS III and CPS I-like enzymes, accompanied with the upregulation of GS and CPS III enzyme proteins in different tissues. Exposure to HEA also led to a sharp rise of plasma cortisol level, suggesting being one of the primary causes of upregulation of GS and CPS III enzymes activity. Liver perfusion experiments further revealed that exposure to HEA enhances the capacity of trapping ammonia to glutamine and urea by the liver of walking catfish. These results suggest that the upregulation of GS and CPS III activity in walking catfish during exposure to HEA plays critical roles to ameliorate the toxic ammonia to glutamine, and also to urea via the induced ornithine-urea cycle possibly through the involvement of cortisol.     

Role of ureogenesis in tackling problems of ammonia toxicity during exposure to higher ambient ammonia in the air-breathing walking catfish<Emphasis Type="Italic">clarias batrachus</Emphasis>

In the present study, the possible role of ureogenesis to avoid the accumulation of toxic ammonia to a lethal level under hyper-ammonia stress was tested in the air-breathing walking catfishClarias batrachus by exposing the fish at 25 mM NH₄Cl for 7 days. Excretion of ammonia by the NH₄Cl-exposed fish was totally suppressed, which was accompanied by significant accumulation of ammonia in different body tissues. The walking catfish, which is otherwise predominantly ammoniotelic, turned totally towards ureotelism from ammoniotelism with a 5-to 6-fold increase of urea-N excretion during exposure to higher ambient ammonia. Stimulation of ureogenesis was accompanied with significant increase of some of the key urea cycle enzymes such as carbamyl phosphate synthetase (urea cycle-related), argininosuccinate synthetase and argininosuccinate lyase both in hepatic and non-hepatic tissues. Due to this unique physiological strategy of turning towards ureotelism from ammoniotelism via the induced urea cycle, this air-breathing catfish is able to survive in very high ambient ammonia, which they face in certain seasons of the year in the natural habitat.     

Elucidating the Strategies for Isolation of Endophytic Fungi and Their Functional Attributes for the Regulation of Plant Growth and Resilience to Stress

Journal of Plant Growth Regulation - Accumulating evidences from investigations in the last decade have revealed the significant role of endophytes in regulation of plant growth, communication and...     

Theoretical studies on the pyridoxal-5'-phosphate dependent enzyme dopa decarboxylase: effect of thr 246 residue on the co-factor-enzyme binding and reaction mechanism

Decarboxylation of amino acid is a key step for biosynthesis of several important cellular metabolites in the biological systems. This process is catalyzed by amino acid decarboxylases and most of them use pyridoxal-5'-phosphate (PLP) as a co-factor. PLP is bound to the active site of the enzyme by various interactions with the neighboring amino acid residues. In the present investigation, density functional theory (DFT) and real-time dynamics studies on both ligand-free and ligand-bound dopa decarboxylases (DDC) have been carried out in order to elucidate the factors responsible for facile decarboxylation and also for proper binding of PLP in the active site of the enzyme. It has been found that in the crystal structure Asp271 interacts with the pyridine nitrogen atom of PLP through H-bonding in both native and substrate-bound DDC. On the contrary, Thr246 is in close proximity to the oxygen of 3-OH ofPLP pyridine ring only in the substrate-bound DDC. In the ligand-free enzyme, the distance between the oxygen atom of 3-OH group of PLP pyridine ring and oxygen atom of Thr246 hydroxyl group is not favorable for hydrogen bonding. Thus, present study reveals that hydrogen bonding with 03 of PLP with a hydrogen bond donor residue provided by the enzyme plays an important role in the decarboxylation process.     

Expression of ornithine-urea cycle enzymes in early life stages of air-breathing walking catfish Clarias batrachus and induction of ureogenesis under hyper-ammonia stress

The air-breathing walking catfish Clarias batrachus is a potential ureogenic teleost with having a full complement of ornithine-urea cycle (OUC) enzymes expressed in various tissues. The present study was aimed at determining the pattern of nitrogenous waste excretion in the form of ammonia-N and urea-N along with the changes of tissue ammonia and urea levels, and the expression of OUC enzymes and glutamine synthetase (GSase) in early life stages of this teleost, and further, to study the possible induction of ureogenesis in 15-day old fry under hyper-ammonia stress. The ammonia and urea excretion was visible within 12 h post-fertilization (hpf), which increased several-fold until the yolk was completely absorbed by the embryo. Although all the early developing stages were primarily ammoniotelic, they also excreted significant amount of nitrogen (N) in the form of urea-N (about 35-40% of total N). Tissue levels of ammonia and urea also increased along with subsequent developmental stages at least until the yolk absorption stage. All the OUC enzymes and GSase were expressed within 4-12 hpf showing an increasing trend of activity for all the enzymes until 350 hpf. There was a significant increase of activity of GSase, carbamyl phosphate synthetase III (CPSase III) and argininosuccinate lyase enzymes (ASL), accompanied with significant increase of enzyme protein concentration of at least two enzymes (GSase and CPSase III) in the 15-day old fry following exposure to 10 mM NH4Cl as compared to respective controls kept in water over a period of 72 h. Thus, it appears that the OUC enzymes are expressed in early life stages of walking catfish like other teleosts, but at relatively high levels and remain expressed all through the life stages with a potential of stimulation of ureogenesis throughout the life cycle as a sort of physiological adaptation to survive and breed successfully under hyper-ammonia and various other environmental-related stresses.     

Mitochondrial citrulline synthesis from ammonia and glutamine in the liver of ureogenic air-breathing catfish, Clarias batrachus (Linnaeus)

The possible synthesis of citrulline, a rate limiting step for urea synthesis via the ornithine-urea cycle (OUC) in teleosts was tested both in the presence of ammonia and glutamine as nitrogen-donating substrates by the isolated liver mitochondria of ureogenic air-breathing walking catfish, C. batrachus. Both ammonia and glutamine could be used as nitrogen-donating substrates for the synthesis of citrulline by the isolated liver mitochondria, since the rate of citrulline synthesis was almost equal in presence of both the substrates. The citrulline synthesis by the isolated liver mitochondria requires succinate at a concentration of 0.1 mM as an energy source, and also requires the involvement of intramitochondrial carbonic anhydrase activity for supplying HCO3 as another substrate for citrulline synthesis. The rate of citrulline synthesis was further stimulated significantly by the isolated liver mitochondria of the fish after pre-exposure to 25 mM NH4Cl for 7 days. Due to possessing this biochemical adaptational strategy leading to the amelioration of ammonia toxicity mainly by channeling ammonia directly and/or via the formation of glutamine to the OUC, this air-breathing catfish could succeed in surviving in high external ammonia, which it faces in its natural habitat in certain seasons of the year.     

Enzymatic Resolution of α‐Methyleneparaconic Acids and Evaluation of their Biological Activity

Both enantiomers of three biologically relevant paraconic acids—MB‐3, methylenolactocin, and C75—were obtained with enantioselectivities up to 99% by kinetic enzymatic resolutions. Good enantiomeric excesses were obtained for MB‐3 and methylenolactocin, using α‐chymotrypsin and aminoacylase as enantiocomplementary enzymes, while C75 was resolved with aminoacylase. They all were evaluated for their antiproliferative, antibacterial, and antifungal activities, showing weak effects and practically no difference between enantiomers in each case. At high concentrations (16–64 µg/mL), (–)‐ C75 acted as an antimicrobial agent against Gram‐positive bacteria. Chirality 27:239–246, 2015. © 2015 Wiley Periodicals, Inc.     

Cell volume changes affect gluconeogenesis in the perfused liver of the catfish<Emphasis Type="Italic">Clarias batrachus</Emphasis>

In addition to lactate and pyruvate, some amino acids were found to serve as potential gluconeogenic substrates in the perfused liver ofClarias batrachus. Glutamate was found to be the most effective substrate, followed by lactate, pyruvate, serine, ornithine, proline, glutamine, glycine, and aspartate. Four gluconeogenic enzymes, namely phosphoenolpyruvate carboxykinase (PEPCK), pyruvate carboxylase (PC), fructose 1,6-bisphosphatase (FBPase) and glucose 6-phosphatase (G6Pase) could be detected mainly in liver and kidney, suggesting that the latter are the two major organs responsible for gluconeogenic activity in this fish. Hypo-osmotically induced cell swelling caused a significant decrease of gluconeogenic efflux accompanied with significant decrease of activities of PEPCK, FBPase and G6Pase enzymes in the perfused liver. Opposing effects were seen in response to hyperosmotically induced cell shrinkage. These changes were partly blocked in the presence of cycloheximide, suggesting that the aniso-osmotic regulations of gluconeogenesis possibly occurs through an inverse regulation of enzyme proteins and/or a regulatory protein synthesis in this catfish. In conclusion, gluconeogenesis appears to play a vital role inC. batrachus in maintaining glucose homeostasis, which is influenced by cell volume changes possibly for proper energy supply under osmotic stress.     

Influence of cell volume changes on autophagic proteolysis in the perfused liver of air-breathing walking catfish (Clarias batrachus)

Exposure of perfused liver of walking catfish (Clarias batrachus) to hypotonicity (-80 mOsmol/L) caused swelling of liver cells as evidenced by the increase in liver mass by 11.5%, and inhibition of [(3)H]leucine release (as a measure of proteolysis) by 37% from the radiolabeled perfused liver. Whereas, exposure of perfused liver to hypertonicity (+80 mOsmol/L) caused shrinkage of liver cells as evidenced by the decrease in liver mass by 10.4%, and stimulation of [(3)H]leucine release by 24%. Infusion of amino acids such as glutamine plus glycine (2 mM each) also caused increase in liver cell volume as evidenced by the increase in liver mass by 8.9%, and inhibition of [(3)H]leucine release by 29%. Adjustment of anisotonicity of the media without changing the NaCl concentration in the media had almost similar effects on proteolysis in the perfused liver. A direct correlation of cell volume changes or hydration status of liver cells with that of proteolysis was observed in the perfused liver regardless of whether the cell volume increase/decrease was evoked by anisotonic perfusion media or by the addition of amino acids. Thus, it appears that the increase/decrease in hepatic cell volume could be one of the important modulators for adjusting the autophagic proteolysis in walking catfish probably to avoid the adverse affects of osmotically induced cell volume changes, to preserve the hepatic cell function and for proper energy supply under osmotic stress. This is the first report of cell volume-sensitive changes of autophagic proteolysis in hepatic cells of any teleosts.