Preparation of magnetic microspheres from water-in-oil emulsion stabilized by block copolymer dispersant

A new method for the preparation of magnetic microspheres is reported. The preparation involved first the dispersion of an aqueous phase, containing magnetite nanoparticles and a water-soluble homopolymer, into droplets in an organic medium using an amphiphilic block copolymer as the dispersant. This was followed by water distillation at a raised temperature from the aqueous droplets to yield polymer/magnetite particles. The structure of the particles was then locked in by a reagent being added to cross-link the water-soluble copolymer block and homopolymer. Since the hydrophobic block of the copolymer consisted of a protected polyester, the removal of the protective moieties from the coronal chains yielded poly(acrylic acid) or other functional polymers to render water dispersibility to the spheres and to enable biomolecule immobilization.     

Aluminum interaction with calmodulin. Evidence for altered structure and function from optical and enzymatic studies

The interaction of aluminum ions with bovine brain calmodulin has been examined by fluorescence spectroscopy, circular dichroic spectrophotometry and equilibrium dialysis, and by the calmodulin-dependent activation of 3',5'-cyclic nucleotide phosphodiesterase. These experiments show that aluminum binds stoichiometrically and cooperatively to calmodulin. Binding of aluminum at a molar ratio of 2:1 to calmodulin suffices to induce a major structural change. Estimates from spectroscopic data indicate that the binding affinity for the first mol of aluminum bound to the protein is about one order of magnitude stronger than that of calcium to its comparable site. These estimates agree with a dissociation constant of 0.4 microM derived from equilibrium dialysis experiments. Interaction of aluminum with calmodulin induces a helix-coil transition and enhances the hydrophobic surface area much more than calcium does. A molar ratio of 4:1 for [aluminum]/[calmodulin] is sufficient to block completely the activity of the calcium-calmodulin-dependent phosphodiesterase. Highly hydrated aluminum ions apparently promote solvent-rich, disordered polypeptide regions in calmodulin which, in turn, profoundly influence the protein's flexibility.     

Large-scale prediction of function shift in protein families with a focus on enzymatic function

Protein function shift can be predicted from sequence comparisons, either using positive selection signals or evolutionary rate estimation. None of the methods have been validated on large datasets, however. Here we investigate existing and novel methods for protein function shift prediction, and benchmark the accuracy against a large dataset of proteins with known enzymatic functions. Function change was predicted between subfamilies by identifying two kinds of sites in a multiple sequence alignment: Conservation-Shifting Sites (CSS), which are conserved in two subfamilies using two different amino acid types, and Rate-Shifting Sites (RSS), which have different evolutionary rates in two subfamilies. CSS were predicted by a new entropy-based method, and RSS using the Rate-Shift program. In principle, the more CSS and RSS between two subfamilies, the more likely a function shift between them. A test dataset was built by extracting subfamilies from Pfam with different EC numbers that belong to the same domain family. Subfamilies were generated automatically using a phylogenetic tree-based program, BETE. The dataset comprised 997 subfamily pairs with four or more members per subfamily. We observed a significant increase in CSS and RSS for subfamily comparisons with different EC numbers compared to cases with same EC numbers. The discrimination was better using RSS than CSS, and was more pronounced for larger families. Combining RSS and CSS by discriminant analysis improved classification accuracy to 71%. The method was applied to the Pfam database and the results are available at http://FunShift.cgb.ki.se. A closer examination of some superfamily comparisons showed that single EC numbers sometimes embody distinct functional classes. Hence, the measured accuracy of function shift is underestimated.     

The organ distribution and circulation time of intravenously injected colloidal carriers sterically stabilized with a block copolymer--poloxamine 908

Colloidal carriers injected intravenously are normally removed rapidly and efficiently by the liver and this represents a major barrier to drug targeting. By coating model microspheres and emulsions with a block co-polymer (poloxamine) it has been possible to keep the carrier circulating in the vascular compartment with little or no uptake by the reticuloendothelial system.     

Cadmium resistance in A549 cells correlates with elevated glutathione content but not antioxidant enzymatic activities

Glutathione has been implicated to function in cytoprotection against cadmium toxicity. The mechanism by which glutathione plays this role has not been well understood. Because glutathione is an important antioxidant and several studies have shown that cadmium induces oxidative stress, this study was undertaken to determine whether development of cadmium resistance is linked to enhanced antioxidant activities. A cadmium-resistant subpopulation of human lung carcinoma A549 cells, which was developed by repeatedly exposing the cells to step-wise increased cadmium concentrations, was compared to a cadmium-sensitive one. The acquired cadmium resistance resulted from neither decreased cadmium uptake nor enhanced cellular metallothionein synthesis. Glutathione content, however, was markedly elevated in the cadmium-resistant cells. In contrast, the activities of the glutathione redox cycle related enzymes, glutathione peroxidase and reductase, were unchanged. Two other antioxidant enzymes, superoxide dismutase and catalase, were also not altered. The results suggest that the development of cadmium resistance in A549 cells unlikely results from enhanced antioxidant enzyme activities, although it is associated with elevated cellular glutathione levels. In addition, measurement of the mRNA and DNA levels for γ-glutamyleysteine synthetase, the rate-limiting enzyme for glutathione biosynthesis, revealed that enhanced expression of the enzyme but not gene amplification is likely responsible for the elevation of cellular glutathione levels.     

HERG is protected from pharmacological block by alpha-1,2-glucosyltransferase function

The HERG (human ether-à-go-go-related gene) protein, which underlies the cardiac repolarizing current I(Kr), is the unintended target for many pharmaceutical agents. Inadvertent block of I(Kr), known as the acquired long QT syndrome (aLQTS), is a leading cause for drug withdrawal by the United States Food and Drug Administration. Hence, an improved understanding of the regulatory factors that protect most individuals from aLQTS is essential for advancing clinical therapeutics in broad areas, from cancer chemotherapy to antipsychotics and antidepressants. Here, we show that the K(+) channel regulatory protein KCR1, which markedly reduces I(Kr) drug sensitivity, protects HERG through glucosyltransferase function. KCR1 and the yeast alpha-1,2-glucosyltransferase ALG10 exhibit sequence homology, and like KCR1, ALG10 diminished HERG block by dofetilide. Inhibition of cellular glycosylation pathways with tunicamycin abrogated the effects of KCR1, as did expression in Lec1 cells (deficient in glycosylation). Moreover, KCR1 complemented the growth defect of an alg10-deficient yeast strain and enhanced glycosylation of an Alg10 substrate in yeast. HERG itself is not the target for KCR1-mediated glycosylation because the dofetilide response of glycosylation-deficient HERG(N598Q) was still modulated by KCR1. Nonetheless, our data indicate that the alpha-1,2-glucosyltransferase function is a key component of the molecular pathway whereby KCR1 diminishes I(Kr) drug response. Incorporation of in vitro data into a computational model indicated that KCR1 expression is protective against arrhythmias. These findings reveal a potential new avenue for targeted prevention of aLQTS.     

Prions: on the enzymatic function of PrPc from mammalian and prion "families"

This minireview deals with some approaches and results of experiments which enabled to discover SOD-like activity of the mammal PrP protein. This activity required the unchanged region of the repeated octapeptide and Cu2+ binding to the appropriate sites of the PrP molecule. It was shown that an infectious prion isoform could bind the normal isoform. This leads to the loss of PrP SOD-like function accompanied by Cu2+ release from the molecule. Also, the problem of sowing the protein seeds of prion propagation is briefly summarized and the first evidence of abnormal protein conformation induced in vivo using yeast cell and in vitro formed Sup 35 prion seed is described.     

Hits, Fhits and Nits: beyond enzymatic function

We have briefly summarized what is known about these proteins, but in closing wish to feature the outstanding questions. Hint1 was discovered mistakenly as an inhibitor of Protein Kinase C and designated Pkci, a designation that still confuses the literature. The other Hint family members were discovered by homology to Hint1. Aprataxin was discovered as a result of the hunt for a gene responsible for AOA1. Fhit was discovered through cloning of a familial chromosome translocation breakpoint on chromosome 3 that interrupts the large FHIT gene within an intron, in the FRA3B chromosome region (Ohta et al., 1996), now known to be the region of the human genome most susceptible to DNA damage due to replication stress (Durkin et al., 2008). The NitFhit fusion genewas discovered during searches for Fhit homologs in flies and worms because the fly/worm Nit polypeptide is fused to the 5'-end of the Fhit gene; the mammalian Nit gene family was discovered because of the NitFhit fusion gene, in searches for homologs to the Nit polypeptide of the NitFhit gene. Each of the Hit family member proteins is reported to have enzymatic activities toward putative substrates involving nucleosides or dinucleosides. Most surprisingly, each of the Hit family proteins discussed has been implicated in important DNA damage response pathways and/or tumor suppression pathways. And for each of them it has been difficult to assign definite substrates, to know if the substrates and catalytic products have biological functions, to know if that function is related to the DNA damage response and suppressor functions, and to precisely define the pathways through which tumor suppression occurs. When the fly Nit sequence was found at the 5'-end of the fly Fhit gene, this gene was hailed as a Rosetta stone gene/protein that would help in discovery of the function of Fhit, because the Nit protein should be in the same signal pathway (Pace et al., 2000). However, the mammalian Nit family proteins have turned out to be at least as mysterious as the Fhit proteins, with the Nit1 substrate still unknown and the surprising finding that Nit proteins also appear to behave as tumor suppressor proteins. Whether the predicted enzymatic functions of these proteins are relevant to the observed biological functions, remain among the outstanding unanswered puzzles and raise the question: have these mammalian proteins evolved beyond the putative original enzymatic purpose, such that the catalytic function is now vestigial and subservient to signal pathways that use the protein-substrate complexes in pathways that signal apoptosis or DNA damage response? Or can these proteins be fulfilling catalytic functions independently but in parallel with signal pathway functions, as perhaps observed for Aprataxin? Or is the catalytic function indeed part of the observed biological functions, such as apoptosis and tumor suppression? Perhaps the recent, post-genomic focus on metabolomics and genome-wide investigations of signal pathway networks will lead to answers to some of these outstanding questions.     

Selective enzymatic degradation of self-assembled particles from amphiphilic block copolymers obtained by the combination of N-carboxyanhydride and nitroxide-mediated polymerization

Combining controlled radical polymerizations and a controlled polypeptide synthetic technique, such as N-carboxyanhydride (NCA) ring-opening polymerization, enables the generation of well-defined block copolymers to be easily accessible. Here we combine NCA polymerization with the nitroxide-mediated radical polymerization of poly(n-butyl acrylate) (PBA) and polystyrene (PS), using a TIPNO and SG1-based bifunctional initiator to create a hybrid block copolymer. The polypeptide block consists of (block) copolymers of poly(L-glutamic acid) embedded with various quantities of L-alanine. The formed superstructures (vesicles and micelles) of the block copolymers possessed varying degrees of enzyme responsiveness when exposed to elastase and thermolysin, resulting in controlled enzymatic degradation dictated by the polypeptide composition. The PBA containing block copolymers possessing 50% L-alanine in the polypeptide block showed a high degradation response compared to polymers containing lower L-alanine quantities. The particles stabilized by copolypeptides with L-alanine near the hydrophobic block showed full degradation within 4 days. Particles containing polystyrene blocks revealed no appreciable degradation under the same conditions, highlighting the specificity of the system and the importance of synthetic polymer selection. However, when the degradation temperature was increased to 70 °C, degradation could be achieved due to the higher block copolymer exchange between the particle and the solution. A number of novel biohybrid structures are disclosed that show promise as enzyme-responsive materials with potential use as payload release vehicles, following their controlled degradation by specific, target, enzymes.     

Intramolecular epistasis and the evolution of a new enzymatic function

Atrazine chlorohydrolase (AtzA) and its close relative melamine deaminase (TriA) differ by just nine amino acid substitutions but have distinct catalytic activities. Together, they offer an informative model system to study the molecular processes that underpin the emergence of new enzymatic function. Here we have constructed the potential evolutionary trajectories between AtzA and TriA, and characterized the catalytic activities and biophysical properties of the intermediates along those trajectories. The order in which the nine amino acid substitutions that separate the enzymes could be introduced to either enzyme, while maintaining significant catalytic activity, was dictated by epistatic interactions, principally between three amino acids within the active site: namely, S331C, N328D and F84L. The mechanistic basis for the epistatic relationships is consistent with a model for the catalytic mechanisms in which protonation is required for hydrolysis of melamine, but not atrazine.     

Insights into structure and function of ionotropic glutamate receptor channels: Starting from channel block

Ionotropic glutamate receptors (iGluRs) are modular proteins that contain ion channel permeable to different cations including calcium. The physiological role of iGluRs is mainly defined by the fact that currents conducted by their channels underlie communication between neurons in the majority of synapses in our brain. Knowing the structure and function of iGluR channel will not only give us a clue to how our brain works but also may help us to develop drugs for the treatment of multiple neurological disorders. Here I give a brief historical overview of the progress made in studies of iGluR channel structure and function that started more than two decades ago with studies of ion channel block. The article is published in the original.     

Molecular structure and enzymatic function of lycopene cyclase from the cyanobacterium Synechococcus sp strain PCC7942.

A gene encoding the enzyme lycopene cyclase in the cyanobacterium Synechococcus sp strain PCC7942 was mapped by genetic complementation, cloned, and sequenced. This gene, which we have named crtL, was expressed in strains of Escherichia coli that were genetically engineered to accumulate the carotenoid precursors lycopene, neurosporene, and zeta-carotene. The crtL gene product converts the acyclic hydrocarbon lycopene into the bicyclic beta-carotene, an essential component of the photosynthetic apparatus in oxygen-evolving organisms and a source of vitamin A in human and animal nutrition. The enzyme also converts neurosporene to the monocyclic beta-zeacarotene but does not cyclize zeta-carotene, indicating that desaturation of the 7-8 or 7'-8' carbon-carbon bond is required for cyclization. The bleaching herbicide 2-(4-methylphenoxy)triethylamine hydrochloride (MPTA) effectively inhibits both cyclization reactions. A mutation that confers resistance to MPTA in Synechococcus sp PCC7942 was identified as a point mutation in the promoter region of crtL. The deduced amino acid sequence of lycopene cyclase specifies a polypeptide of 411 amino acids with a molecular weight of 46,125 and a pI of 6.0. An amino acid sequence motif indicative of FAD utilization is located at the N terminus of the polypeptide. DNA gel blot hybridization analysis indicated a single copy of crtL in Synechococcus sp PCC7942. Other than the FAD binding motif, the predicted amino acid sequence of the cyanobacterial lycopene cyclase bears little resemblance to the two known lycopene cyclase enzymes from nonphotosynthetic bacteria. Preliminary results from DNA gel blot hybridization experiments suggest that, like two earlier genes in the pathway, the Synechococcus gene encoding lycopene cyclase is homologous to plant and algal genes encoding this enzyme.     

The elevated homocysteine stimulates changes of haemostatic function of plasma isolated from breast cancer patients

The aim of our study was to explain the effect of elevated homocysteine (measured by HPLC) on haemostatic activity of plasma from breast cancer patients (fibrin polymerization and lysis; the thrombin and prothrombin time), because homocysteine (Hcys) induces changes in haemostasis, as well blood clotting as fibrinolysis. Patients were hospitalized in Department of Oncological Surgery, Medical University of Lodz, Poland. All patients have not had preadjuvant therapy, and samples from patients were taken before surgery. We observed that changes of selected parameters of haemostatic properties of plasma, e.g., the prothrombin time and thrombin time were prolonged in plasma from invasive breast cancer when compared with the control group (healthy subjects) and patients with benign breast diseases. Our results showed also that the correlation between the increased amount of Hcys and changes of selected parameters of haemostasis in invasive breast cancer patients exists. Considering the data presented in this study, we suggest that the elevated Hcys in invasive breast cancer patients may induce the changes of haemostatic properties of plasma isolated from these patients.     

Commonly stabilized cytochromes c from deep-sea Shewanella and Pseudomonas

Two cytochromes c₅ (SBcytc and SVcytc) have been derived from Shewanella living in the deep-sea, which is a high pressure environment, so it could be that these proteins are more stable at high pressure than at atmospheric pressure, 0.1 MPa. This study, however, revealed that SBcytc and SVcytc were more stable at 0.1 MPa than at higher pressure. In addition, at 0.1–150 MPa, the stability of SBcytc and SVcytc was higher than that of homologues from atmospheric-pressure Shewanella, which was due to hydrogen bond formation with the heme in the former two proteins. This study further revealed that cytochrome c₅₅₁ (PMcytc) of deep-sea Pseudomonas was more stable than a homologue of atmospheric-pressure Pseudomonas aeruginosa, and that specific hydrogen bond formation with the heme also occurred in the former. Although SBcytc and SVcytc, and PMcytc are phylogenetically very distant, these deep-sea cytochromes c are commonly stabilized through hydrogen bond formation.     

Self-assembled monolayer of light-harvesting core complexes from photosynthetic bacteria on a gold electrode modified with alkanethiols

Light-harvesting antenna core (LH1-RC) complexes isolated from Rhodoseudomonas palustris were self-assembled on a gold electrode modified with self-assembled monolayers (SAMs) of the alkanethiols NH2(CH2)nSH, n = 2, 6, 8, 11; HOOC(CH2)7SH; and CH3(CH2)7SH, respectively. Adsorption of the LH1-RC complexes on the SAMs depended on the terminating group of the alkanethiols, where the adsoption increased in the following order for the terminating groups: amino groups > carboxylic acid groups > methyl groups. Further, the adsorption on a gold electrode modified with SAMs of NH2(CH2)nSH, n = 2, 6, 8, 11, depended on the methylene chain length, where the adsorption increased with increasing the methylene chain length. The presence of the well-known light-harvesting and reaction center peaks of the near infrared (NIR) absorption spectra of the LH1-RC complexes indicated that these complexes were only fully stable on the SAM gold electrodes modified with the amino group. In the case of modification with the carboxyl group, the complexes were partially stable, while in the presence of the terminal methyl group the complexes were extensively denatured. An efficient photocurrent response of these complexes on the SAMs of NH2(CH2)nSH, n = 2, 6, 8, 11, was observed upon illumination at 880 nm. The photocurrent depended on the methylene chain length (n), where the maximum photocurrent response was observed at n = 6, which corresponds to a distance between the amino terminal group in NH2(CH2)6SH and the gold surface of 1.0 nm.     

Monomer of sodium and potassium ion activated adenosinetriphosphatase displays complete enzymatic function

The distribution of sodium and potassium ion activated adenosinetriphosphatase [(Na+ + K+)-ATPase] among the various oligomeric forms present in a given solution is assessed unambiguously by cross-linking with glutaraldehyde. Purified enzyme dissolved in a solution of a nonionic detergent, octaethylene glycol dodecyl ether, remains dispersed and unaggregated after removal of the bulk of the detergent. Increases in the aggregation of the enzyme, which have been previously observed upon the addition of substrates to such a solution, are found to be due to changes in ionic strength rather than a consequence of the initiation of turnover. Furthermore, conditions are described that produce solutions containing stable, enzymatically active mixtures of the smaller oligomers of the asymmetric unit, alpha beta. Cross-linking by glutaraldehyde while the enzyme is turning over demonstrates that at least one of these oligomers is responsible for the observed enzymatic activity. A determination of which oligomers are present in each fraction from a glycerol gradient demonstrates that the profiles of the enzymatic activity and the concentration of monomer coincide. In addition, the monomer can form the sodium-dependent, phosphorylated intermediate of the mechanism for the enzyme. Finally, a preparation of (Na+ + K+)-ATPase, dissolved in solutions of the same nonionic detergent, can be prepared in which the predominant species (greater than 85%) is the monomer. The enzyme in this solution exhibits high specific activity, and its apparent Michaelis constants for the cationic substrates are very similar to those of the purified, membrane-bound enzyme. It is concluded from these results that a monomer of the alpha beta asymmetric unit is fully capable of catalyzing (Na+ + K+)-ATPase activity, and hence active transport, in the native enzyme. A reassessment of proposed molecular mechanisms for active transport is made in light of these discoveries.