Water relations, activities of antioxidants, ethylene evolution and membrane integrity of pigeonpea roots as affected by soil moisture

The plants of pigeonpea (Cajanus cajan L.) cv. H77-216 were subjected to moderate [soil moisture content (SMC) = 7.3 ± 0.5 %] and severe (SMC = 4.3 ± 0.5 %) drought by withholding the irrigation at vegetative stage (45 d after sowing). The control plants were maintained at SMC of 11.0 ± 0.5 %. Half of the stressed plants were re-irrigated and their recovery was studied after 2 d. Leaf water potential, osmotic potential, and relative water content of leaf and root decreased significantly while a sharp rise in proline and total soluble sugars contents were noticed. Drought induced a significant increase in 1-aminocyclopropane 1-carboxylic acid (ACC) content and ACC oxidase activity which caused a considerable increase in ethylene evolution. Malondialdehyde content and relative stress injury were increased under drought whereas reverse was true for ascorbic acid content. The membrane integrity of roots decreased during stress and recovered on rehydration. The specific activity of total superoxide dismutase, ascorbate peroxidase, glutathione reductase, and glutathione transferase decreased to 37 – 78 %, 17 – 62 %, 29 – 36 % and 57 – 79 % at moderate and severe drought, respectively. The increase in activity of catalase and peroxidase could not overcome the accumulation of H₂O₂ content in the roots.     

Role of phenylalanine B10 in plant nonsymbiotic hemoglobins

All plants contain an unusual class of hemoglobins that display bis-histidyl coordination yet are able to bind exogenous ligands such as oxygen. Structurally homologous hexacoordinate hemoglobins (hxHbs) are also found in animals (neuroglobin and cytoglobin) and some cyanobacteria, where they are thought to play a role in free radical scavenging or ligand sensing. The plant hxHbs can be distinguished from the others because they are only weakly hexcacoordinate in the ferrous state, yet no structural mechanism for regulating hexacoordination has been articulated to account for this behavior. Plant hxHbs contain a conserved Phe at position B10 (Phe(B10)), which is near the reversibly coordinated distal His(E7). We have investigated the effects of Phe(B10) mutation on kinetic and equilibrium constants for hexacoordination and exogenous ligand binding in the ferrous and ferric oxidation states. Kinetic and equilibrium constants for hexacoordination and ligand binding along with CO-FTIR spectroscopy, midpoint reduction potentials, and the crystal structures of two key mutant proteins (F40W and F40L) reveal that Phe(B10) is an important regulatory element in hexacoordination. We show that Phe at this position is the only amino acid that facilitates stable oxygen binding to the ferrous Hb and the only one that promotes ligand binding in the ferric oxidation states. This work presents a structural mechanism for regulating reversible intramolecular coordination in plant hxHbs.     

Molecular Identification of Tropical Tasar Silkworm (Antheraea mylitta) Ecoraces with RAPD and SCAR Markers

The tropical tasar silkworm, Antheraea mylitta, has several ecoraces, 10 of which are commercially exploited for the production of tasar silk. These ecoraces are identified by morphological markers that are greatly influenced by photoperiod, humidity, altitude, and host plants. The DNA markers, random amplification of polymorphic DNA (RAPD), and sequence-characterized amplified region (SCAR) are identified to complement the existing morphological markers. Seven RAPD bands are selected that identify 8 of the 10 ecoraces. These identified RAPD fragments are sequenced and primers are designed for SCAR markers. Of the seven sets of primers, a single primer pair produced polymorphic SCAR bands that diagnose 5 of the 10 ecoraces. All 10 ecoraces are identified by the use of RAPD and SCAR markers together.     

Hyperthermophilic asparaginase mutants with enhanced substrate affinity and antineoplastic activity: structural insights on their mechanism of action

Thermophilic l-asparaginases display high stability and activity at elevated temperatures. However, they are of limited use in leukemia therapy because of their low substrate affinity and reduced activity under physiological conditions. In an attempt to combine stability with activity at physiological conditions, 3 active-site mutants of Pyrococcus furiosus l-asparaginase (PfA) were developed. The mutants, specifically K274E, showed improved enzymatic properties at physiological conditions as compared to the wild type. All variants were thermodynamically stable and resistant to proteolytic digestion. None of the enzymes displayed glutaminase activity, a highly desirable therapeutic property. All variants showed higher and significant killing of human cell lines HL60, MCF7, and K562 as compared to the Escherichia coli l-asparaginase. Our study revealed that increased substrate accessibility through the active site loop plays a major role in determining activity. A new mechanistic insight has been proposed based on molecular dynamics simulated structures, where dynamic flipping of a critical Tyr residue is responsible for the activity of thermophilic l-asparaginases. Our study not only resulted in development of PfA mutants with combination of desirable properties but also gave a mechanistic insight about their activity.     

Increased endogenous H2S generation by CBS, CSE, and 3MST gene therapy improves ex vivo renovascular relaxation in hyperhomocysteinemia

Hydrogen sulfide (H(2)S) has recently been identified as a regulator of various physiological events, including vasodilation, angiogenesis, antiapoptotic, and cellular signaling. Endogenously, H(2)S is produced as a metabolite of homocysteine (Hcy) by cystathionine β-synthase (CBS), cystathionine γ-lyase (CSE), and 3-mercaptopyruvate sulfurtransferase (3MST). Although Hcy is recognized as vascular risk factor at an elevated level [hyperhomocysteinemia (HHcy)] and contributes to vascular injury leading to renovascular dysfunction, the exact mechanism is unclear. The goal of the current study was to investigate whether conversion of Hcy to H(2)S improves renovascular function. Ex vivo renal artery culture with CBS, CSE, and 3MST triple gene therapy generated more H(2)S in the presence of Hcy, and these arteries were more responsive to endothelial-dependent vasodilation compared with nontransfected arteries treated with high Hcy. Cross section of triple gene-delivered renal arteries immunostaining suggested increased expression of CD31 and VEGF and diminished expression of the antiangiogenic factor endostatin. In vitro endothelial cell culture demonstrated increased mitophagy during high levels of Hcy and was mitigated by triple gene delivery. Also, dephosphorylated Akt and phosphorylated FoxO3 in HHcy were reversed by H(2)S or triple gene delivery. Upregulated matrix metalloproteinases-13 and downregulated tissue inhibitor of metalloproteinase-1 in HHcy were normalized by overexpression of triple genes. Together, these results suggest that H(2)S plays a key role in renovasculopathy during HHcy and is mediated through Akt/FoxO3 pathways. We conclude that conversion of Hcy to H(2)S by CBS, CSE, or 3MST triple gene therapy improves renovascular function in HHcy.     

Undersulfation of heparan sulfate restricts differentiation potential of mouse embryonic stem cells

Heparan sulfate proteoglycans, present on cell surfaces and in the extracellular matrix, interact with growth factors and morphogens to influence growth and differentiation of cells. The sulfation pattern of the heparan sulfate chains formed during biosynthesis in the Golgi compartment will determine the interaction potential of the proteoglycan. The glucosaminyl N-deacetylase/N-sulfotransferase (NDST) enzymes have a key role during biosynthesis, greatly influencing total sulfation of the heparan sulfate chains. The differentiation potential of mouse embryonic stem cells lacking both NDST1 and NDST2 was studied using in vitro differentiation protocols, expression of differentiation markers, and assessment of the ability of the cells to respond to growth factors. The results show that NDST1 and NDST2 are dispensable for mesodermal differentiation into osteoblasts but necessary for induction of adipocytes and neural cells. Gene expression analysis suggested a differentiation block at the primitive ectoderm stage. Also, GATA4, a primitive endoderm marker, was expressed by these cells. The addition of FGF4 or FGF2 together with heparin rescued the differentiation potential to neural progenitors and further to mature neurons and glia. Our results suggest that the embryonic stem cells lacking both NDST1 and NDST2, expressing a very low sulfated heparan sulfate, can take the initial step toward differentiation into all three germ layers. Except for their potential for mesodermal differentiation into osteoblasts, the cells are then arrested in a primitive ectoderm and/or endoderm stage.     

Tracking viral evolution during a disease outbreak: the rapid and complete selective sweep of a circovirus in the endangered Echo parakeet

Circoviruses are among the smallest and simplest of all viruses, but they are relatively poorly characterized. Here, we intensively sampled two sympatric parrot populations from Mauritius over a period of 11 years and screened for the circovirus Beak and feather disease virus (BFDV). During the sampling period, a severe outbreak of psittacine beak and feather disease, which is caused by BFDV, occurred in Echo parakeets. Consequently, this data set presents an ideal system for studying the evolution of a pathogen in a natural population and to understand the adaptive changes that cause outbreaks. Unexpectedly, we discovered that the outbreak was most likely caused by changes in functionally important regions of the normally conserved replication-associated protein gene and not the immunogenic capsid. Moreover, these mutations were completely fixed in the Echo parakeet host population very shortly after the outbreak. Several capsid alleles were linked to the replication-associated protein outbreak allele, suggesting that whereas the key changes occurred in the latter, the scope of the outbreak and the selective sweep may have been influenced by positive selection in the capsid. We found evidence for viral transmission between the two host populations though evidence for the invasive species as the source of the outbreak was equivocal. Finally, the high evolutionary rate that we estimated shows how rapidly new variation can arise in BFDV and is consistent with recent results from other small single-stranded DNA viruses.     

Scaffold hybridization in generation of indenoindolones as anticancer agents that induce apoptosis with cell cycle arrest at G2/M phase

Scaffold hybridization of several natural and synthetic anticancer leads led to the consideration of indenoindolones as potential novel anticancer agents. A series of these compounds were prepared by a diversity-feasible synthetic method. They were found to possess anticancer activities with higher potency compared to etoposide and 5-fluorouracil in kidney cancer cells (HEK 293) and low toxicity to corresponding normal cells (Vero). They exerted apoptotic effect with blocking of cell cycle at G2/M phase.     

Evaluation of immunostimulatory and growth promoting effect of seed fractions of Achyranthes aspera in common carp Cyprinus carpio and identification of active constituents

Immunostimulatory and growth promoting properties of Achyranthes aspera seeds were studied with larvae of common carp Cyprinus carpio. Four experimental diets were prepared using raw (D1) and alcohol (D2), petroleum ether (D3) and 50% aqueous alcohol (D4) extracts of A. aspera seeds. Diet without seed served as control (D5). Fish were fed with test/control diet for 30 days and then immunized with 10 μl of c-RBC. Blood samples were collected 7 days after immunization. Survival (93 ± 3%) of fish was significantly (P < 0.05) higher in D1 diet fed group compared to others. Highest specific growth rate was found in fish fed with diet D2. Significantly (P < 0.05) higher levels of serum protein and albumin were found in D1 and D3 compared to others. Highest serum globulin level was found in D1, which was followed by D3, D2, D4 and D5. Hemagglutination titer level was 5-18 folds higher in diet D3 fed fish compared to others. SGOT and SGPT levels were significantly (P < 0.05) higher in control group compared to the treated groups. Myeloperoxidase activity was significantly (P < 0.05) higher in D1 (2.513 ± 0.27 λ 450 nm) and D3 (2.38 ± 0.07 λ 450 nm) diets fed groups compared to others. The best performance of fish was found in raw A. aspera seeds incorporated diet fed group and the active constituents were identified as ecdysterone and two essential fatty acids linolenic acid and oleic acid.