Studies on the purification of banana polyphenoloxidase

Studies on the extractability of polyphenoloxidase (PPO) from the pulp of five banana cultivars revealed a varietal difference in the nature of binding of the PPO in the cell, with the enzyme being entirely in the soluble fraction in one and partly associated with the cell wall in others, necessitating use of a detergent to release it from the latter. Partial purification by acetone precipitation and chromatography using a DEAE-cellulose column yielded two major fractions DE-I and DE-II with purifications of 4- and 16·3-fold and activity recoveries of 38·2 and 43·3% respectively. Further gel filtration of the two fractions on a Sephadex G-100 column improved the purifications to 44- and 50-fold respectively with full activity recovery. Polyacrylamide gel electrophoretic studies showed the two fractions to be composed of isoenzymes differing in pattern. The purified enzyme showed maximum absorption at 275 nm.

Pulsed laser surface treatment of magnesium alloy: Correlation between thermal model and experimental observations

The effect of deposition of Al +Al2O3 on MRI 153 M Mg alloy processed using a pulsed Nd:YAG laser is presented in this study. A composite coating with metallurgical joint to the substrate was formed. The microstructure and phase constituents were characterized and correlated with the thermal predictions. The laser scan speed had an effect on the average melt depth and the amount of retained and/or reconstituted alumina in the final coating. The coating consisted of alumina particles and highly refined dendrites formed due to the extremely high cooling rates (of the order of 10(8) K/s). The microhardness of the coating was higher and several fold improvement of wear resistance compared to the substrate was observed for the coatings. These microstructural features and physical properties were correlated with the effects predicted by a thermal model.

Kinetics of interaction of Cotton Leaf Curl Kokhran Virus-Dabawali (CLCuKV-Dab) coat protein and its mutants with ssDNA

Gemini viral assembly and transport of viral DNA into nucleus for replication, ssentially involve DNA-coat protein interactions. The kinetics of interaction of Cotton LeafCtirl Kokhran Virus-Dabawali recombinant coat protein (rCP) with DNA was studied by electrophoretic mobility shift assay (EMSA) and Surface plasmon resonance (SPR). The rCP interacted with ssDNA with a K-A, of 2.6 +/- 0.29 x 10(8) M-1 in a sequence non-specific manner. The CP has a conserved C2H2 type zinc finger motif composed of residues C68, C72, H81 and H85. Mutation of these residues to alanine resulted in reduced binding to DNA probes. The H85A mutant rCP showed the least binding with approximately 756 fold loss in the association rate and a three order magnitude decrease in the binding affinity as compared to rCP. The CP-DNA interactions via the zinc finger motif could play a Crucial role ill Virus assembly and in nuclear transport. (C) 2009 Elsevier Inc.

Crystal and molecular structure of the quinoxaline antibiotic analog TANDEM (des-N-tetramethyltriostin A)

The crystal structure of TANDEM (des-N-tetramethyltriostin A), a synthetic analogue of the quinoxaline antibiotic triostin A, has been determined independently at -135 and 7 'C and refined to R values of 0.088 and 0.147, respectively. The molecule has approximate 2-fold symmetry, with the quinoxaline chromophores and the disulfide cross-bridge projecting from opposite sides of the peptide ring. The quinoxaline groups are nearly parallel to each other and separated by about 6.5 A. The peptide backbone resembles a distorted antiparallel 13 ribbon joined by intramolecular hydrogen bonds N-H(LVal)--O(L-Ala). At low temperatures, the TANDEM molecule is surrounded by a regular first- and second-order hydration sphere containing 14 independent water molecules. At room temperature, only the first-order hydration shell is maintained. Calculations of the interplanar separation of the quinoxaline groups as a function of their orientation with respect to the peptide ring support the viability of TANDEM to intercalate bifunctionally into DNA.

Low-frequency dielectric constant measurements in the critical polar + non-polar binary liquid system: Methanol + n-heptane

The low-frequency (5–100 kHz) dielectric constant ε has been measured in the temperature range 7 × 10−5 < T = (T − Tc)/Tc < 8 × 10−2. Near Tc an exponent ≈0.11 characterizes the power law behaviour of dε/dt consistent with the theoretically predicted t−α singularity. However, over the full range of t an exponent ≈0.35 is obtained.

Cholesterol-Esterifying Enzymes in Developing Rat Brain

A cholesterol-esterifying enzyme which incorporates exogenous fatty acids into cholesterol esters in the presence of ATP and coenzyme A was demonstrated in 15-day-old rat brain. This enzyme was maximally active at pH 7.4 and distinct from the cholesterol-esterifying enzyme reported earlier (Eto and Suzuki, 1971), which has a pH optimum at 5.2 and does not require cofactors. Properties of the two enzymes have been compared. Both the enzymes showed negligible esterification with acetate and were maximally active with oleic acid. The pH 5.2 enzyme esterified desmosterol, lanosterol and cholesterol at about the same rate, while the pH 7.4 enzyme was only 50% as active ith lanosterol as it was with cholesterol and desmosterol. Phosphatidyl serine stimulated the pH 5.2 enzyme but not the pH 7.4 enzyme. Phosphatidyl choline and sodium taurocholate showed no effect on either of the enzymes. Both the enzymes were associated with particulate fractions, but the pH 7.4 enzyme was localized more in the microsomes. Purified myelin showed 2.6-fold and 1.5-fold higher specific activities of pH 5.2 and 7.4 enzymes respectively, when compared with homogenate. About 7-10% of total activity of both the enzymes was associated with purified myelin. Brain stem and spinal cord showed higher specific activity of pH 5.2 enzyme than cerebral cortex and cerebellum, while pH 7.4 enzyme specific activity was higher in cerebellum and brain stem than in cerebral cortex and spinal cord. Microsomal pH 7.4 activity showed progressive increase prior to the active period of myelination, reaching a maximum on the 15th day after birth and declined to 20% of the peak activity by 30 days. In contrast, pH 5.2 enzyme reached maximum activity about the 6th day after birth and remained at this level well into adulthood. In 15-day-old rat brain, pH 7.4 enzyme had five to six times higher specific activity than pH 5.2 enzyme, while in adults the activities were equal. The pH 7.4 enzyme showed a threefold higher specific activity than pH 5.2 enzyme in myelin from 15-day-old rats, but in adults the reverse was true. Key Words: Cholesterol esterifying enzymes-Developing rat brain-Myelination. Jagannatha H. M. and Sastry P. S. Cholesterol-esterifying enzymes in developing rat brain. J. Neurochem. 36, 1352- 1360 (1981).

Foldable compatibility matrix for the folding of programmable logic arrays

This paper describes a switching theoretic algorithm for the folding of programmable logic arrays (PLA). The algorithm is valid for both column and row folding, although it has been presented considering only the simple column folding. The pairwise compatibility relations among all the pairs of the columns of the PLA are mapped into a square matrix, called the compatibility matrix of the PLA. A foldable compatibility matrix (FCM), a new concept introduced by the author, is then derived from the compatibility matrix. A new theorem called the folding theorem is then proved. The theorem states that the existence of an m by 2m FCM is both necessary and sufficient to fold 2m columns of the n column PLA (2m ≤ n). Once an FCM is obtained, the ordered pairs of foldable columns and the re-ordering of the rows are readily determined.

A Novel Cache Architecture and Placement Framework for Packet Forwarding Engines

Packet forwarding is a memory-intensive application requiring multiple accesses through a trie structure. With the requirement to process packets at line rates, high-performance routers need to forward millions of packets every second with each packet needing up to seven memory accesses. Earlier work shows that a single cache for the nodes of a trie can reduce the number of external memory accesses. It is observed that the locality characteristics of the level-one nodes of a trie are significantly different from those of lower level nodes. Hence, we propose a heterogeneously segmented cache architecture (HSCA) which uses separate caches for level-one and lower level nodes, each with carefully chosen sizes. Besides reducing misses, segmenting the cache allows us to focus on optimizing the more frequently accessed level-one node segment. We find that due to the nonuniform distribution of nodes among cache sets, the level-one nodes cache is susceptible t high conflict misses. We reduce conflict misses by introducing a novel two-level mapping-based cache placement framework. We also propose an elegant way to fit the modified placement function into the cache organization with minimal increase in access time. Further, we propose an attribute preserving trace generation methodology which emulates real traces and can generate traces with varying locality. Performanc results reveal that our HSCA scheme results in a 32 percent speedup in average memory access time over a unified nodes cache. Also, HSC outperforms IHARC, a cache for lookup results, with as high as a 10-fold speedup in average memory access time. Two-level mappin further enhances the performance of the base HSCA by up to 13 percent leading to an overall improvement of up to 40 percent over the unified scheme.

Estrogen Induction Of Biotin-Binding Protein In Immature Chicks - Kinetics, Hormonal Specificity And Modulation

Employing a specific radioimmunoassay for quantification, the kinetics of estrogen-induced elevation in the plasma concentration of biotin-binding protein (BBP) in immature male chicks was investigated. A single injection of the steroid hormone enhanced the plasma BBP content several-fold at 6 h, reaching peak levels around 48 h and declining thereafter. A 2-fold amplification of the response was evident during secondary stimulation with the hormone. The magnitude of the response was hormonal dose-dependent while the initial lag phase and the time of peak protein accumulation were unaltered within the hormonal doses tested. The circulatory half-life of the specific protein in normal and estrogenized birds was 10 h. Hyperthyroidism markedly decreased the hormonal response while the opposite effect was seen during hypothyroidism. The antiestrogens E- and Z-clomiphene citrate effectively blocked the protein induction whereas progesterone, either alone or in combination with estrogen, was ineffective in modulating the induction. Cycloheximide administration drastically inhibited the inductive response. The above observations clearly suggest that the genes corresponding to the two isofunctional proteins of chicken egg, viz. BBP and avidin, are differentially regulated.

Three-dimensional elastic analysis of cracked thick plates under bending fields

A three-dimensional analysis is presented for the bending problem of finite thick plates with through-the-thickness cracks. A general solution is obtained for Navier's equations of the theory of elasticity. It is found that the in-plane stresses and the transverse normal stress at the crack front are singular with an inverse square root singularity, while the transverse shear stresses are of the order of unity. Results from a numerical study indicate that the stress intensity factor, which varies across the thickness, is influenced by the thickness ratio in a significant manner. Results from a parametric study and those from a comparative study with existing finite element values are presented.

Binding of N-dansylgalactosamine to winged-bean tuber lectin: studies by fluorescence quenching titrations

The winged-bean tuber lectin binds to N-dansyl(5-dimethylaminonaphthalene-1-sulphonic acid)galactosamine, leading to a 12.5-fold increase in dansyl fluorescence with a concomitant 25 nm blue-shift in the emission maximum. The enhancement of fluorescence intensity was completely reversed by the addition of methyl α-galactopyranoside. The lectin has two binding sites per molecule for this fluorescent sugar and an association constant of 2.59 · 105 M−1 at 25° C. The binding of N-dansylgalactosamine to the lectin shows that it can accommodate a large hydrophobic substituent on the C-2 carbon of d-galactose. Studies with other sugars indicate that a hydrophobic substituent with α-conformation at the anomeric position increases the affinity of binding. The C-4 and C-6 hydroxyl groups are also critical for sugar binding to this lectin.

Modulation of arginine decarboxylase activity in cucumber (Cucumis sativus) cotyledons in short-term organ culture

Among the various amines administered to excisedCucumis sativus cotyledons in short-term organ culture, agmatine (AGM) inhibited arginine decarboxylase (ADC) activity to around 50%, and putrescine was the most potent entity in this regard. Homoarginine (HARG) dramatically stimulated (3- to 4-fold) the enzyme activity. Both AGM inhibition and HARG stimulation of ADC were transient, the maximum response being elicited at 12 h of culture. Mixing experiments ruled out involvement of a macromolecular effector in the observed modulation of ADC. HARG-stimulated ADC activity was completely abolished by cycloheximide, whereas AGM-mediated inhibition was unaffected. Half-life of the enzyme did not alter on treatment with either HARG or AGM. The observed alterations in ADC activity are accompanied by change in Km of the enzyme. HARG-stimulated ADC activity is additive to that induced by benzyladenine (BA) whereas in presence of KCl, HARG failed to enhance ADC activity, thus demonstrating the overriding influence of K+ on amine metabolism.

Stimulation of liver tryptophan pyrrolase during heat exposure

Exposure of rats to heat (39 +/- 1 degree C) stimulated liver tryptophan pyrrolase 2-fold between 3 and 48 h. Plasma corticosterone increased 2-fold after 1 h of heat exposure and decreased to a low value of 50% by 16 h. The effect of heat exposure on the enzyme was obtained in adrenalectomized animals. Stimulation by cortisol and tryptophan of the enzyme was also obtained in heat exposure, and the effects seemed to be additive. The concentration of tryptophan in the liver remained unchanged, and that in the plasma decreased to about 50% at 8 h exposure to heat and reverted to normal by 46 h. Simultaneous administration of noradrenaline to heat-exposed rats had no effect, whereas that of thyroxine partly prevented the stimulation of the enzyme activity. Hypothyroid conditions obtained by thyroidectomy or treatment with propylthiouracil significantly stimulated the enzyme activity. Cycloheximide treatment of heat-exposed rats did not prevent the stimulation of the enzyme activity. The results indicate that the effect of heat exposure on liver tryptophan pyrrolase is obtained, due to the accompanying hypothyroid condition, by increasing the activity of the existing protein by a mechanism possibly different from those known at present.

Structural effects of a dimer interface mutation on catalytic activity of triosephosphate isomerase.The role of conserved residues and complementary mutations

The active site of triosephosphate isomerase (TIM, EC: 5.3.1.1), a dimeric enzyme, lies very close to the subunit interface. Attempts to engineer monomeric enzymes have yielded well-folded proteins with dramatically reduced activity. The role of dimer interface residues in the stability and activity of the Plasmodium falciparum enzyme, PfTIM, has been probed by analysis of mutational effects at residue 74. The PfTIM triple mutant W11F/W168F/Y74W (Y74W*) has been shown to dissociate at low protein concentrations, and exhibits considerably reduced stability in the presence of denaturants, urea and guanidinium chloride. The Y74W* mutant exhibits concentration-dependent activity, with an approximately 22-fold enhancement of kcat over a concentration range of 2.5–40 μm, suggesting that dimerization is obligatory for enzyme activity. The Y74W* mutant shows an approximately 20-fold reduction in activity compared to the control enzyme (PfTIM WT*, W11F/W168F). Careful inspection of the available crystal structures of the enzyme, together with 412 unique protein sequences, revealed the importance of conserved residues in the vicinity of the active site that serve to position the functional K12 residue. The network of key interactions spans the interacting subunits. The Y74W* mutation can perturb orientations of the active site residues, due to steric clashes with proximal aromatic residues in PfTIM. The available crystal structures of the enzyme from Giardia lamblia, which contains a Trp residue at the structurally equivalent position, establishes the need for complementary mutations and maintenance of weak interactions in order to accommodate the bulky side chain and preserve active site integrity.

Aspects of tautomerism 9-solvolysis of normal and pseudo phthaloyl chlorides

Solvolysis of normal and pseudo phthaloyl chlorides in aqueous acetone and in aqueous dioxane has revealed that the former solvolyses about hundred-fold faster than the latter. Contrary to the accepted belief, there is no evidence for equilibrium between the normal and the pseudo forms of phthaloyl chloride, under a variety of conditions.