Electrostatic factors in DNA intercalation

The factors that determine the binding of a chromophore between the base pairs in DNA intercalation complexes are dissected. The electrostatic potential in the intercalation plane is calculated using an accurate ab initio based distributed multipole electrostatic model for a range of intercalation sites, involving different sequences of base pairs and relative twist angles. There will be a significant electrostatic contribution to the binding energy for chromophores with a predominantly positive electrostatic potential, but this varies significantly with sequence, and somewhat with twist angle. The usefulness of these potential maps for understanding the binding of intercalators is explored by calculating the electrostatic binding energy for 9-aminoacridine, ethidium, and daunomycin in a variety of model binding sites. The electrostatic forces play a major role in the positioning of an intercalating 9-aminoacridine and a significant stabilizing role in the binding of ethidium in its sterically constrained position, but the intercalation of daunomycin is determined by the side-chain binding. Sequence preferences are likely to be determined by a complex and subtle mixture of effects, with electrostatics being just one component. The electrostatic binding energy is also unlikely to be a major determinant of the twist angle, as its variation with angle is modest for most intercalation sites. Overall, the electrostatic potential maps give guidance on how positively charged chromophores can be chemically adapted by heteroatomic substitution to optimise their binding.     

Solid-state fermentation in rotating drum bioreactors: operating variables affect performance through their effects on transport phenomena

Aspergillus oryzae ACM 4996 was grown on an artificial gel-based substrate and on steamed wheat bran during solid-state fermentations in 18.7 L rotating drum bioreactors. For gel fermentations fungal growth decreased as rotational speed increased, presumably due to increased shear. For wheat bran fermentations fungal growth improved under agitated compared to static culture conditions, due to superior heat and mass transfer. We conclude that the effects of operational variables on the performance of SSF bioreactors are mediated by their effects on transport phenomena such as mixing, shear, heat transfer, and mass transfer within the substrate bed. In addition, the substrate characteristics affect the need for and the rates of these transport processes. Different transport phenomena may be rate limiting with different substrates. This work improves understanding of the effects of bioreactor operation on SSF performance.     

Effect of dietary fish on antioxidant parameters of normal and cholesterol stressed rats

Feeding fish (Sardinella longiceps) to normal rats increased lipid peroxidation and total and Se-dependent glutathione peroxidase (GSH-px) activity in erythrocytes and manganese dependent superoxide dismutase (Mn-SOD) activity in liver. Feeding fish to cholesterol stressed rats showed a significant increase in the activity of GSH-px and cholesterol feeding alone, resulted in a significant increase in the lipid peroxidation and liver Mn-SOD activity. The results suggest that the high polyunsaturated fatty acid content of S. longiceps, the fish abundantly available in the west coast of India, does not have any deleterious effect by way of free radical generation. The observed lipid peroxidation is not critical as is evident from the results of glutathione level and other scavenging enzymes.     

Expansion of haematopoietic stem cells in vitro: a challenge to stem cell biologists

Expansion of haematopoietic stem cells from various sources has gained importance so as to provide a clinically potential graft, which shows ideal growth kinetics, resulting in reduction of the period of neutropenia and thrombocytopenia in any autologous or allogenic transplant setting. Expansion also facilitates transduction of genes for gene therapy. This review examines the various means employed to achieve the expansion of stem cells, and the criteria used to score the extent of expansion based on how stem cells are identified. It tries to analyse the ideal manner in which expansion should be carried out, with emphasis that expansion should not be at the expense of loss of stemness. It also attempts to judge the roles played by the stromal elements and cytokines, which are both part of the complex microenvironment, which in vivo has a strict regulation on haematopoiesis.     

Regulation of ProMMP-1 and ProMMP-3 activation by tissue factor pathway inhibitor-2/matrix-associated serine protease inhibitor

Tissue factor pathway inhibitor-2 (TFPI-2)/matrix-associated serine protease inhibitor (MSPI), a 32- to 33-kDa Kunitz-type serine protease inhibitor, inhibits plasmin and trypsin. Because plasmin and trypsin are involved in the activation of promatrix metalloproteases proMMP-1 and proMMP-3, we investigated the role of TFPI-2/MSPI in the activation of these proenzymes. Both plasmin and trypsin activated proMMP-1 by converting the 53-kDa proenzyme to the partially active 43-kDa polypeptide; this activity was inhibited by TFPI-2/MSPI. Similarly, TFPI-2/MSPI inhibited the conversion of 66-kDa proMMP-3 to the activated 45- and 30-kDa polypeptides by plasmin and trypsin. Because plasmin is involved in the physiological activation of proMMP-3, we tested whether TFPI-2/MSPI inhibits the activation of proMMP-3 by HT-1080 fibrosarcoma cells and urokinase-charged HeLa cells. We found that the inhibitor inhibited proMMP-3 activation by HT-1080 cells and urokinase-charged HeLa cells. Collectively, our results suggest that TFPI-2/MSPI indirectly regulates MMP-1- and MMP-3-catalyzed matrix proteolysis by regulating the activation of proMMP-1 and proMMP-3.     

The flattened face of type II beta phosphatidylinositol phosphate kinase binds acidic phospholipid membranes

Type II beta phosphatidylinositol phosphate kinase is a representative phosphatidylinositol phosphate kinase that is active against membrane-bound substrates. The structure of the enzyme contains a flattened basic face that spans the crystallographic dimer interface and is adjacent to the active site. Analytical ultracentrifugation shows that phosphatidylinositol phosphate kinase is a dimer in solution. Modeling suggested that the flattened face binds to acidic phospholipids by electrostatic interactions. The enzyme binds to acidic vesicles containing phosphatidylserine, phosphatidic acid, or phosphoinositides mixed with phosphatidylcholine, but not to neutral phosphatidylcholine vesicles. Binding to acidic vesicles is abolished in the presence of 1.0 M NaCl, consistent with an essential electrostatic contribution to the free energy of binding. The +14 charge on the flattened face of the dimer was reduced to +2 in the triple mutant Lys72Glu/Lys76Glu/Lys78Glu. The mutation has no effect on dimerization, but reduces the apparent KA for 25% phosphatidylserine/75% phosphatidylcholine mixed vesicles by 16-fold. The reduction in the level of binding can be ascribed to a loss of electrostatic interactions based on the finite difference solution to the Poisson-Boltzmann equation. The mutant reduces catalytic activity toward phosphatidylinositol 5-phosphate by approximately 50-fold. The wild-type enzyme binds half-maximally to phosphatidylinositol 4,5-bisphosphate-containing vesicles at a mole fraction of 0.3% in a phosphatidylcholine background, as compared to a 22% mole fraction in phosphatidylserine. The binding to phosphatidylinositol 4,5-bisphosphate-containing membranes is less sensitive to salt and to the triple mutation than binding to phosphatidylserine-containing membranes, suggesting that at least part of phosphatidylinositol 4,5-bisphosphate's interaction with the enzyme is independent of the flattened face. It is concluded that the flattened face of type II beta phosphatidylinositol phosphate kinase binds to membranes through nonspecific interactions, and that this interaction is essential for efficient catalysis.     

Sources of stream sulfate at the Hubbard Brook Experimental Forest: Long-term analyses using stable isotopes

Sulfur deposition in the northeastern U.S. has been decreasing since the 1970s and there has been a concomitant decrease in the SO42– lost from drainage waters from forest catchments of this region. It has been established previously that the SO42– lost from drainage waters exceeds SO42– inputs in bulk precipitation, but the cause for this imbalance has not been resolved. The use of stable S isotopes and the availability of archived bulk precipitation and stream water samples at the Hubbard Brook Experimental Forest (HBEF) in New Hampshire provided a unique opportunity to evaluate potential sources and sinks of S by analyzing the long-term patterns (1966–1994) of the 34S values of SO42–. In bulk precipitation adjacent to the Ecosystem Laboratory and near Watershed 6 the 34S values were greater (mean: 4.5 and 4.2l, respectively) and showed more variation (variance: 0.49 and 0.30) than stream samples from Watersheds 5 (W5) and 6 (W6) (mean: 3.2 and 3.7j; variance: 0.09 and 0.08, respectively). These results are consistent with other studies in forest catchments that have combined results for mass balances with stable S isotopes. These results indicate that for those sites, including the HBEF, where atmospheric inputs are 10 kg S ha–1 yr–1, most of the deposited SO42– cycles through the biomass before it is released to stream water. Results from W5, which had a whole-tree harvest in 1983–1984 showed that adsorption/desorption processes play an important role in regulating net SO42– retention for this watershed-ecosystem. Although the isotopic results suggest the importance of S mineralization, conclusive evidence that there is net mineralization has not yet been shown. However, S mass balances and the isotopic result are consistent with the mineralization of organic S being a major contributor to the SO42– in stream waters at the HBEF.     

Conformation and stability of barley chymotrypsin inhibitor-2 (CI-2) mutants containing multiple lysine substitutions

A major goal of agricultural biotechnology is to increase the nutritional value of maize seed through the expression of heterologous proteins enriched in lysine. One promising candidate is barley chymotrypsin inhibitor-2 (CI-2), a plant protein that has been extensively characterized with respect to structure and function. Based on the tertiary structure of wild-type (WT) CI-2, five mutants with lysine contents ranging from 20 to 25 mol percent were designed, expressed in Escherichia coli and purified by ion exchange and gel permeation chromatography. Inasmuch as previous transgenic experiments suggested that proper folding and stability may be essential for in vivo accumulation of the engineered proteins in plant cells, we first undertook an in vitro study of the conformation and thermodynamic stability of the CI-2 mutants in order to select an ideal candidate for plant expression. Mutant and WT CI-2 proteins had similar circular dichroism spectra, suggesting similar secondary structures. However, differences in the accessibility of the sole tryptophan residue, Trp24, indicated that the local conformation differed among the mutants. The thermodynamic stability of the mutants ranged from <2 to 4.9 kcal/mol compared with approximately 7 kcal/mol for the wild-type protein. In conjunction with proteolytic stability studies, we have identified one mutant that has the potential to be expressed in a stable manner in plant cells.