Homology and enzymatic requirements of microhomology-dependent alternative end joining

Nonhomologous DNA end joining (NHEJ) is one of the major double-strand break (DSB) repair pathways in higher eukaryotes. Recently, it has been shown that alternative NHEJ (A-NHEJ) occurs in the absence of classical NHEJ and is implicated in chromosomal translocations leading to cancer. In the present study, we have developed a novel biochemical assay system utilizing DSBs flanked by varying lengths of microhomology to study microhomology-mediated alternative end joining (MMEJ). We show that MMEJ can operate in normal cells, when microhomology is present, irrespective of occurrence of robust classical NHEJ. Length of the microhomology determines the efficiency of MMEJ, 5 nt being obligatory. Using this biochemical approach, we show that products obtained are due to MMEJ, which is dependent on MRE11, NBS1, LIGASE III, XRCC1, FEN1 and PARP1. Thus, we define the enzymatic machinery and microhomology requirements of alternative NHEJ using a well-defined biochemical system.

Non-enzymatic electronic detection of glucose using aminophenylboronic acid functionalized reduced graphene oxide

We report the non-enzymatic electronic detection of glucose using field effect transistor (FET) devices made of aminophenylboronic acid (APBA) functionalized reduced graphene oxide (RGO). Detection of glucose molecules was carried out over a wide dynamic range of concentration varying from 100 pM to 100 mM with a detection limit of similar to 2 nM using both covalently and non-covalently functionalized APBA-RGO complex. The normalized change in electrical conductance data shows that the FET devices made of non-covalently functionalized APBA-RGO complex (nc-APBA-RGO) exhibited a linear response to glucose aqueous solution of concentrations varying from 1 nM to 10 mM and showed 4 times enhanced sensitivity over the devices made of covalently functionalized APBA-RGO complex (c-APBA-RGO). Specificity of APBA-RGO complex to glucose is confirmed from the observation of negligible change in electrical conductance after exposure to 0.1 mM of lactose and other interfering factors. (C) 2015 Elsevier B.V. All rights reserved.

Enzymatic degradation of polycaprolactone-gelatin blend

Blends of polycaprolactone (PCL), a synthetic polymer and gelatin, natural polymer offer a optimal combination of strength, water wettability and cytocompatibility for use as a resorbable biomaterial. The enzymatic degradation of PCL, gelatin and PCL-gelatin blended films was studied in the presence of lipase (Novozym 435, immobilized) and lysozyme. Novozym 435 degraded the PCL films whereas lysozyme degraded the gelatin. Though Novozym 435 and lysozyme individually could degrade PCL-gelatin blended films, the combination of these enzymes showed the highest degradation of these blended films. Moreover, the enzymatic degradation was much faster when fresh enzymes were added at regular intervals. The changes in physico-chemical properties of polymer films due to degradation were studied by scanning electron microscopy, Fourier transform infrared spectroscopy and differential scanning calorimetry. These results have important implications for designing resorbable biomedical implants.

Enzymatic degradation of polycaprolactone-gelatin blend

Blends of polycaprolactone (PCL), a synthetic polymer and gelatin, natural polymer offer a optimal combination of strength, water wettability and cytocompatibility for use as a resorbable biomaterial. The enzymatic degradation of PCL, gelatin and PCL-gelatin blended films was studied in the presence of lipase (Novozym 435, immobilized) and lysozyme. Novozym 435 degraded the PCL films whereas lysozyme degraded the gelatin. Though Novozym 435 and lysozyme individually could degrade PCL-gelatin blended films, the combination of these enzymes showed the highest degradation of these blended films. Moreover, the enzymatic degradation was much faster when fresh enzymes were added at regular intervals. The changes in physico-chemical properties of polymer films due to degradation were studied by scanning electron microscopy, Fourier transform infrared spectroscopy and differential scanning calorimetry. These results have important implications for designing resorbable biomedical implants.

Characterisation of cell adhesion to substrate materials and the resistance to enzymatic and mechanical cell-removal

Cell-implant adhesive strength is important for prostheses. In this paper, an investigation is described into the adhesion of bovine chondrocytes to Ti6Al4V-based substrates with different surface roughnesses and compositions. Cells were cultured for 2 or 5 days, to promote adhesion. The ease of cell removal was characterised, using both biochemical (trypsin) and mechanical (accelerated buoyancy and liquid flow) methods. Computational fluid dynamics (CFD) modelling has been used to estimate the shear forces applied to the cells by the liquid flow. A comparison is presented between the ease of cell detachment indicated using these methods, for the three surfaces investigated. © 2008 Materials Research Society.

Mechanistic studies of a model reaction for enzymatic hydroxylation

A study has been made of the reaction mechanism of a model system for enzymatic hydroxylation. The results of a kinetic study of the hydroxylation of 2-hydroxyazobenzene derivatives by cupric ion and hydrogen peroxide are presented. An investigation of kinetic orders indicates that hydroxylation proceeds by way of a coordinated intermediate complex consisting of cupric ion and the mono anions of 2-hydroxyazobenzene and hydrogen peroxide. Studies with deuterated substrate showed the absence of a primary kinetic isotope effect and no evidence of an NIH shift. The effect of substituents on the formation of intermediate complexes and the overall rate of hydroxylation was studied quantitatively in aqueous solution. The combined results indicate that the hydroxylation step is only slightly influenced by ring substitution. The substituent effect is interpreted in terms of reaction by a radical path or a concerted mechanism in which the formation of ionic intermediates is avoided. The reaction mechanism is discussed as a model for enzymatic hydroxylation.

Heterometallic Complexes as Models of Enzymatic Active Sites

This dissertation describes studies on two multinucleating ligand architectures: the first scaffold was designed to support tricopper complexes, while the second platform was developed to support tri- and tetrametallic clusters.

In Chapter 2, the synthesis of yttrium (and lanthanide) complexes supported by a tripodal ligand framework designed to bind three copper centers in close proximity is described. Tricopper complexes were shown to react with dioxygen in a 1:1 [Cu₃]/O₂ stoichiometry to form intermediates in which the O–O bond was fully cleaved, as characterized via UV-Vis spectroscopy and determination of the reaction stoichiometry. Pre-arrangement of the three Cu centers was pivotal to cooperative O₂ activation, as mono-copper complexes reacted differently with dioxgyen. The reactivity of the observed intermediates was studied with various substrates (reductants, O-atom acceptors, H-atom donors, Brønsted acids) to determine their properties. In Chapter 3, the reactivity of the same yttrium-tricopper complex with nitric oxide was explored. Reductive coupling to form a trans-hyponitrite complex (characterized by X-ray crystallography) was observed via cooperative reactivity by an yttrium and a copper center on two distinct tetrametallic units. The hyponitrite complex was observed to release nitrous oxide upon treatment with a Brønsted acid, supporting its viability as an intermediate in nitric oxide reduction to nitrous oxide.

In Chapter 4, a different multinucleating ligand scaffold was employed to synthesize heterometallic triiron clusters containing one oxide and one hydroxide bridges. The effects of the redox-inactive, Lewis acidic heterometals on redox potential was studied by cyclic voltammetry, unveiling a linear correlation between redox potential and heterometal Lewis acidity. Further studies on these complexes showed that the Lewis acidity of the redox-inactive metals also affected the oxygen-atom transfer reactivity of these clusters. Comparisons of this reactivity with manganese systems, collaborative efforts to reassign the structures of related manganese oxo-hydroxo clusters, and synthetic attempts to access related dioxo clusters are also described.

In Appendix A, ongoing efforts to synthesize new clusters supported by the same multinucleating ligand platform are described. Studies of novel approaches towards ligand exchange in tetrametallic clusters and incorporation of new supporting and bridging ligand motifs in trinuclear complexes are presented.

Changes of enzymatic activity during larval development of carp, Catla catla (Ham.)

Quantitative assays of trypsin, amylase and alkaline phosphatases were made in relation to age and food during the larval development of the Indian major carp Catla catla. The responses of all the test enzymes to age and food were identical. No enzymes were detected from the fertilized eggs. Detectable amount of enzymes were first observed in the first day old hatchlings. All the test enzymes in the group fed normal feed tended to rise gradually with advancement of age till day 22 after which an asymptotic level was attained. Absence of food throughout the rearing period caused the enzymatic activity of the larva to remain at the lowest level throughout. When starvation was followed by feeding, enzymatic activity in the former group was consistently higher than that of latter, suggesting that feeding activity was primarily responsible in maintaining the enzymatic activity of carp larva. The enzymatic activity of zooplankton was significantly higher than carp larva till day 6 to 12 after which the latter exceeded the former implying that carp larva during development utilizes the exogenous enzymes of zooplankton.