Organic solvent tolerance of an alkaline protease from salt-tolerant alkaliphilic <Emphasis Type="Italic">Streptomyces clavuligerus</Emphasis> strain Mit-1

A salt-tolerant alkaliphilic actinomycete, Mit-1 was isolated from Mithapur, coastal region of Gujarat, India. The strain was identified as Streptomyces clavuligerus and based on 16S rRNA gene sequence (EU146061) homology; it was related to Streptomyces sp. (AY641538.1). The organism could grow with up to 15% salt and pH 11, optimally at 5% and pH 9. It was able to tolerate and secrete alkaline protease in the presence of a number of organic solvents including xylene, ethanol, acetone, butanol, benzene and chloroform. Besides, it could also utilize these solvents as the sole source of carbon with significant enzyme production. However, the organism produced spongy cell mass with all solvents and an orange brown soluble pigment was evident with benzene and xylene. Further, the enzyme secretion increased by 50-fold in the presence of butanol. With acetone and ethanol; the enzyme was highly active at 60–80°C and displayed optimum activity at 70°C. The protease was significantly stable and catalyzed the reaction in the presence of xylene, acetone and butanol. However, ethanol and benzene affected the catalysis of the enzyme adversely. Crude enzyme preparation was more stable at 37°C in solvents as compared to partially purified and purified enzymes. The study holds significance as only few salt-tolerant alkaliphilic actinomycetes are explored and information on their enzymatic potential is still scares. To the best of our knowledge this is the first report on organic solvent tolerant protease from salt-tolerant alkaliphilic actinomycetes.     

Molecular Characterization of a Genomic Fragment Containing Pi-kh Gene from the Genomic Library of Indica Rice Line Tetep

Identification of full length genes along with upstream regulatory elements is important to understand its expression. Here, we report preparation of high titre genomic library and identification of a genomic clone containing Pi-k ^h gene with its complete upstream and downstream sequences from the rice blast resistant line Tetep. Structural analysis of protein revealed that Pi-k ^h has a central nucleotide binding site domain, leucine-rich repeats domain and a unique zinc-finger domain. Comparative analysis of Pi-k ^h protein sequence showed 64% and 45% similarity with the protein sequences of rice blast resistance genes Pi-b and Pi-ta , respectively.     

The encapsulation technology in fruit plants—A review

Encapsulation technology is an exciting and rapidly growing area of biotechnological research. This has drawn tremendous attention in recent years because of its wide use in conservation and delivery of tissue cultured plants of commercial and economic importance. Production of synthetic seeds by encapsulating somatic embryos, shoot buds or any other meristmatic tissue helps in minimizing the cost of micropropagated plantlets for commercialization and final delivery. In most of fruit crops, seed propagation has not been successful because of heterozygosity of seeds, minute seed size, presence of reduced endosperm, low germination rate, and also some are having seedless varieties. Many species have desiccation-sensitive intermediate or recalcitrant seeds and can be stored for only a few weeks or months. Under these circumstances, increasing interest has been shown recently to use encapsulation technology for propagation and conservation. Many fruit plants are studied worldwide for breeding, genetic engineering, propagation, and pharmaceutical purposes. In this context, synthetic seeds would be more applicable in exchange of elite and axenic plant material between laboratories and extension centers due to small bead size and ease in handling. Due to these advantages, interest in using encapsulation technology has continuously been increasing in several fruit plant species. The purpose of this review is to focus upon current information on development of synthetic seeds in several fruit crops.     

Functional Rescue of Mutant Human Cystathionine ��-Synthase by Manipulation of Hsp26 and Hsp70 Levels in Saccharomyces cerevisiae

Many human diseases are caused by missense substitutions that result in misfolded proteins that lack biological function. Here we express a mutant form of the human cystathionine β-synthase protein, I278T, in Saccharomyces cerevisiae and show that it is possible to dramatically restore protein stability and enzymatic function by manipulation of the cellular chaperone environment. We demonstrate that Hsp70 and Hsp26 bind specifically to I278T but that these chaperones have opposite biological effects. Ethanol treatment induces Hsp70 and causes increased activity and steady-state levels of I278T. Deletion of the SSA2 gene, which encodes a cytoplasmic isoform of Hsp70, eliminates the ability of ethanol to restore function, indicating that Hsp70 plays a positive role in proper I278T folding. In contrast, deletion of HSP26 results in increased I278T protein and activity, whereas overexpression of Hsp26 results in reduced I278T protein. The Hsp26-I278T complex is degraded via a ubiquitin/proteosome-dependent mechanism. Based on these results we propose a novel model in which the ratio of Hsp70 and Hsp26 determines whether misfolded proteins will either be refolded or degraded.Cells have evolved quality control systems for misfolded proteins, consisting of molecular chaperones (heat shock proteins) and proteases. These molecules help prevent misfolding and aggregation by either promoting refolding or by degrading misfolded protein molecules (1). In eukaryotic cells, the Hsp70 system plays a critical role in mediating protein folding. Hsp70 protein interacts with misfolded polypeptides along with co-chaperones and promotes refolding by repeated cycles of binding and release requiring the hydrolysis of ATP (2). Small heat shock proteins (sHsp)² are small molecular weight chaperones that bind non-native proteins in an oligomeric complex and whose function is poorly understood (3). In mammalian cells, the sHsp family includes the α-crystallins, whose orthologue in Saccharomyces cerevisiae is Hsp26. Studies suggest that Hsp26 binding to misfolded protein aggregates is a prerequisite for effective disaggregation and refolding by Hsp70 and Hsp104 (4, 5).Misfolded proteins can result from missense substitutions such as those found in a variety of recessive genetic diseases, including cystathionine β-synthase (CBS) deficiency. CBS is a key enzyme in the trans-sulfuration pathway that converts homocysteine to cysteine (6). Individuals with CBS deficiency have extremely elevated levels of plasma total homocysteine, resulting in a variety of symptoms, including dislocated lenses, osteoporosis, mental retardation, and a greatly increased risk of thrombosis (7). Approximately 80% of the mutations found in CBS-deficient patients are point mutations that are predicted to cause missense substitutions in the CBS protein (8). The most common mutation found in CBS-deficient patients, an isoleucine to threonine substitution at amino acid position 278 (I278T), has been observed in nearly one-quarter of all CBS-deficient patients. Based on the crystal structure of the catalytic core of CBS, this mutation is located in a β-sheet more than 10 Å distant from the catalytic pyridoxal phosphate and does not directly affect the catalytic binding pocket or the dimer interface (9).Previously, our lab has developed a yeast bioassay for human CBS in which yeast expressing functional human CBS can grow in media lacking cysteine, whereas yeast expressing mutant CBS cannot (10). We have used this assay to characterize the functional effects of many different CBS missense alleles, including I278T (7, 11). However, an unexpected finding was that it was possible to restore function to I278T and a number of other CBS missense mutations by either truncation or the addition of a second missense mutation in the C-terminal regulatory domain (12, 13). The ability to restore function by a cis-acting second mutation suggested to us that it might be possible to restore function in trans via either interaction of mutant CBS with a small molecule (i.e. drug) or a mutation in another yeast gene. In a previous study, we found that small osmolyte chemical chaperones could restore function to mutant CBS presumably by directly stabilizing the mutant CBS protein (14).In this study we report on the surprising finding that exposure of yeast to ethanol can restore function of I278T CBS by altering the ratio of the molecular chaperones Hsp26 and Hsp70. We demonstrate Hsp70 binding promotes I278T folding and activity, whereas Hsp26 binding promotes I278T degradation via the proteosome. By manipulating the levels of Hsp26 and Hsp70, we are able to show that I278T CBS protein can have enzymatic activity restored to near wild-type levels of activity. Our findings suggest a novel function for sHsps.     

Aluminium(III), chromium(III) and iron(III) complexes with 5-iodouracil and 5-iodouracil-histidine and their antitumour activity

Complexes of the type [Al(HL)(OH)Cl(2)], [M(HL)(OH)(2)Cl] and [M'(HL)(L')(OH)Cl], where HL = 5-iodouracil; HL' = histidine; M = Cr(III), Fe(III) and M' = Al(III), Cr(III), Fe(III), were synthesized and characterized. The complexes are polymeric showing high decomposition points and are insoluble in water and common organic solvents. The mu(eff) values, electronic spectral bands and ESR spectra suggest a polymeric 6-coordinate spin-free octahedral stereochemistry for the Cr(III) and Fe(III) complexes. 5-Iodouracil acts as a monodentate ligand coordinating to the metal ion through the O atom of C((4)) = O while histidine through the O atom of -COO(- ) and the N atom of -NH(2) group. In vivo antitumour effect of 5-iodouracil and its complexes was examined on C(3)H /He mice against P815 murine mastocytoma. As evident from their T/C values, Cr(III) and Fe(III) complexes display significant and higher antitumour activity compared to the 5-iodouracil ligand. The in vitro results of the complexes on the same cells indicate that Cr(III) and Fe(III) complexes show higher inhibition on (3)H-thymidine and (3)H-uridine incorporation in DNA and RNA replication, respectively, at a dose of 5 microg/mL.     

Structural characterization of CalO2: a putative orsellinic acid P450 oxidase in the calicheamicin biosynthetic pathway

Although bacterial iterative Type I polyketide synthases are now known to participate in the biosynthesis of a small set of diverse natural products, the subsequent downstream modification of the resulting polyketide products remains poorly understood. Toward this goal, we report the X-ray structure determination at 2.5 A resolution and preliminary characterization of the putative orsellenic acid P450 oxidase (CalO2) involved in calicheamicin biosynthesis. These studies represent the first crystal structure for a P450 involved in modifying a bacterial iterative Type I polyketide product and suggest the CalO2-catalyzed step may occur after CalO3-catalyzed iodination and may also require a coenzyme A- (CoA) or acyl carrier protein- (ACP) bound substrate. Docking studies also reveal a putative docking site within CalO2 for the CLM orsellinic acid synthase (CalO5) ACP domain which involves a well-ordered helix along the CalO2 active site cavity that is unique compared with other P450 structures.     

Altered Immunohistochemical Expression of Mast Cell Tryptase and Chymase in the Pathogenesis of Oral Submucous Fibrosis and Malignant Transformation of the Overlying Epithelium

Mast cells (MCs) expressing serine proteases; tryptase and chymase, are associated with fibrosis in various diseases. However, little is known about their involvement in oral submucous fibrosis (OSF). Our goal was to evaluate the role of MC tryptase and chymase in the pathogenesis of OSF and its malignant transformation. Immunohistochemical expression of MC tryptase and chymase was evaluated in 20 cases of OSF, 10 cases of oral squamous cell carcinoma (OSCC) and 10 cases of healthy controls. Subepithelial zone of Stage 1 and 2 while deep zone of Stage 3 and 4 OSF demonstrated increased tryptase positive MCs. OSCC revealed a proportionate increase in tryptase and chymase positive MCs irrespective of areas of distribution. An altered balance in the subepithelial and deep distribution of tryptase and chymase positive MCs play an important role in the pathogenesis of OSF and its malignant transformation.     

Simultaneous Over-Expression of PaSOD and RaAPX in Transgenic Arabidopsis thaliana Confers Cold Stress Tolerance through Increase in Vascular Lignifications

Antioxidant enzymes play a significant role in eliminating toxic levels of reactive oxygen species (ROS), generated during stress from living cells. In the present study, two different antioxidant enzymes namely copper-zinc superoxide dismutase derived from Potentilla astrisanguinea (PaSOD) and ascorbate peroxidase (RaAPX) from Rheum austral both of which are high altitude cold niche area plants of Himalaya were cloned and simultaneously over-expressed in Arabidopsis thaliana to alleviate cold stress. It was found that the transgenic plants over-expressing both the genes were more tolerant to cold stress than either of the single gene expressing transgenic plants during growth and development. In both single (PaSOD, RaAPX) and double (PaSOD + RaAPX) transgenic plants higher levels of total antioxidant enzyme activities, chlorophyll content, total soluble sugars, proline content and lower levels of ROS, ion leakage were recorded when compared to the WT during cold stress (4°C), besides increase in yield. In the present study, Confocal and SEM analysis in conjunction with qPCR data on the expression pattern of lignin biosynthetic pathway genes revealed that the cold stress tolerance of the transgenic plants might be because of the peroxide induced up-regulation of lignin by antioxidant genes mediated triggering.