Drug-metal interactions: copper(II) complex of the antidiabetic drug acetohexamide and pyridine

The preparation, spectral properties, and crystal structure of a copper(II) complex of 4-acetyl-N-[(cyclohexylamino)carbonyl]benzenesulfonamide, which is known as acetohexamide, and pyridine are reported. The complex Cu(AH)2(py)2, where AH = acetohexamide and py = pyridine, was prepared and characterized by X-ray and ESR. The complex is monoclinic, space group P2(1)/a, with a = 17.412(6), b = 9.039(2), c = 26.531(10) A, beta = 102.24(2) degrees, and Z = 4. The final refinement used 3892 unique reflections and gave an R value of 0.0646. The copper atom is surrounded by four nitrogen atoms in a square-planar arrangement, two from the acetohexamide ligands (Cu-N = 2.009 A) and two from the pyridine molecules (Cu-N = 2.016 A) in a trans geometry. The ESR data support a similar coordination behavior of the copper (g parallel greater than g perpendicular greater than ge) with the unpaired electron in the dx2-y2 orbital.     

Generalized Baum-Welch Algorithm Based on the Similarity between Sequences

The profile hidden Markov model (PHMM) is widely used to assign the protein sequences to their respective families. A major limitation of a PHMM is the assumption that given states the observations (amino acids) are independent. To overcome this limitation, the dependency between amino acids in a multiple sequence alignment (MSA) which is the representative of a PHMM can be appended to the PHMM. Due to the fact that with a MSA, the sequences of amino acids are biologically related, the one-by-one dependency between two amino acids can be considered. In other words, based on the MSA, the dependency between an amino acid and its corresponding amino acid located above can be combined with the PHMM. For this purpose, the new emission probability matrix which considers the one-by-one dependencies between amino acids is constructed. The parameters of a PHMM are of two types; transition and emission probabilities which are usually estimated using an EM algorithm called the Baum-Welch algorithm. We have generalized the Baum-Welch algorithm using similarity emission matrix constructed by integrating the new emission probability matrix with the common emission probability matrix. Then, the performance of similarity emission is discussed by applying it to the top twenty protein families in the Pfam database. We show that using the similarity emission in the Baum-Welch algorithm significantly outperforms the common Baum-Welch algorithm in the task of assigning protein sequences to protein families.     

Vitamin E, membrane fluidity, and blood pressure in hypertensive and normotensive rats

Vitamin E treatment was found to lower blood pressure, and increase membrane fluidity in rats. The objectives of this study were to investigate the effects of the antioxidant, vitamin E, on the blood pressure and erythrocyte membrane fluidity in spontaneously hypertensive (SHR) and normotensive (WKY) rats. Membrane fluidity was assessed using spin labeling technique and electron paramagnetic resonance (EPR) spectroscopy. Two different spin labels were used in this study, 5-doxylstrearic acid (5-SASL) and 16-doxylstearic acid (16-SASL). The rats were given vitamin E, 3 days/week for 3 weeks and blood pressure was measured once weekly, using the tail-cuff method. Subsequently, blood was taken via heart puncture and erythrocytes were prepared for spin labeling. The fluidity of the membrane in the nonpolar region of erythrocytes from hypertensive rats was found quite different from that of normal rats as judged by the spectra of 16-SASL. The values of maximum splitting parameter of the EPR spectra of the spin label 5-SASL incorporated in erythrocyte membrane from both SHR and WKY rats, and the effects of vitamin E on membrane fluidity were compared. The maximum splitting parameter calculated from EPR spectra was larger for SHR than WKY rats. Additionally, the maximum splitting parameter calculated for vitamin E treated SHR and WKY rats were lower than those of their respective controls. As expected, the blood pressure of the SHR rats was found to be higher than that of the WKY rats. Vitamin E treated SHR and WKY rats showed significantly lower blood pressure than their controls.     

Cell‐free production of a therapeutic protein: Expression,purification, and characterization of recombinant streptokinase using a CHO lysate

The use of cell‐free systems to produce recombinant proteins has grown rapidly over the past decade. In particular, cell‐free protein synthesis (CFPS) systems based on mammalian cells provide alternative methods for the production of many proteins, including those that contain disulfide bonds, glycosylation, and complex structures such as monoclonal antibodies. In the present study, we show robust production of turbo green fluorescent protein (tGFP) and streptokinase in a cell‐free system using instrumented mini‐bioreactors for highly reproducible protein production. We achieved recombinant protein production (～600 μg/ml of tGFP and 500 μg/ml streptokinase) in 2.5 hr of expression time, comparable to previously reported yields for cell‐free protein expression. Also, we demonstrate the use of two different affinity tags for product capture and compare those to a tag‐free self‐cleaving intein capture technology. The intein purification method provided a product recovery of 86%, compared with 52% for conventionally tagged proteins, while resulting in a 30% increase in total units of activity of purified recombinant streptokinase compared with conventionally tagged proteins. These promising beneficial features combined with the intein technology makes feasible the development of dose‐level production of therapeutic proteins at the point‐of‐care.     

Drug-metal interactions: spectroscopic studies of copper hydantoin complexes.

The preparation and spectral properties of copper(II) complexes of two hydantoins are reported. Complexes of the general formula Cu(hyd)2(py)2, where hyd = phenytoin or nirvanol; and py = pyridine were prepared and characterized by infrared and ESR. Spectral data show that the copper atom is bound to the nitrogen atom of the hydantoin anion and to the nitrogen atom of the pyridine molecule to form 2:2:1 hydantoin:pyridine:copper complexes. The ESR data indicate that both complexes have tetragonal symmetry (g11 greater than g perpendicular greater than g e) with the unpaired electron in the d x2-y2 orbital.     

A distance‐dependent atomic knowledge‐based potential and force for discrimination of native structures from decoys

The purpose of this article is to introduce a novel model for discriminating correctly folded proteins from well designed decoy structures using mechanical interatomic forces. In our model, we consider a protein as a collection of springs and the force imposed to each atom is calculated. A potential function is obtained from statistical contact preferences within known protein structures. Combining this function with the spring equation, the interatomic forces are calculated. Finally, we consider a structure and define a score function on the 3D structure of a protein. We compare the force imposed to each atom of a protein with the corresponding atom in the other structures. We then assign larger scores to those atoms with lower forces. The total score is the sum of partial scores of atoms. The optimal structure is assumed to be the one with the highest score in the data set. To evaluate the performance of our model, we apply it on several decoy sets. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Separation of membranes from semiprotoplasts of suspension-cultured sycamore maple (Acer pseudoplatanus) cells

Semiprotoplasts were produced from suspension-cultured Acer pseudoplatanus (sycamore maple) cells prior to cell disruption by passing them through a 60 μm nylon screen. Cell membranes from homogenates were separated by ultracentrifugation on linear sucrose density gradients. Samples were collected by gradient fractionation and subcellular fractions were assayed for membrane markers and glycosyl transferase activities. Results of standard marker assays (cytochrome c reductase for endoplas-mic reticulum. uridine and inosine diphosphatases for Golgi. and eosin-5'-maleimide binding for plasma membrane) showed partial separation of these three membrane types. Golgi and plasma membrane markers overlapped in most gradients. Incorporation of ¹⁴C-labeled sugars from UDP-glucose and UDP-xylose into ethanol precipitated polysaccharides was used to detect glucan synthases I & II (glucosyl transferases) and xylosyl transferase activities in Golgi membrane fractions. All three glycosyl transferases overlapped in fractions corresponding to both Golgi and plasma membrane markers, although peak activities for all three occurred in different fractions. More than one peak of glucan synthase I activity was found. Glucan synthase II, associated with ß-l.3 glucan (cullose) synthesis in plasma membranes, was also detected and exhibited a 10-fold stimulation in the presence of Ca²⁺.     

Characterization of a mononuclear copper carboxylate complex: Bis(acetylsalicylato)bis(pyridine)copper(II)

The preparation, spectral properties, and crystal structure of a mononuclear copper(II) complex of acetylsalicylate and pyridine are reported. The complex exists as bis(acetylsalicylato)bis(pyridine)copper(II) both in the solid state and in chloroform solution. The crystal is monoclinic, space group P2₁/n, with a = 17.823(5), b = 10.903(4), c = 6.598(2) Å, β = 95.74(2)°. The final refinement used 1472 observed reflections and gave an R of 0.046. The copper atom is surrounded by four atoms in a trans square planar arrangement with two short CuO distances of 1.949(3) Å and two CuN distances of 2.003(4) Å. Two longer CuO distances of 2.623(3) Å are made with the remaining oxygen atoms of the aspirin carboxylate groups.     

Spectroscopic and biological studies of spin-labeled tetracycline.

A new nitroxyl labeled tetracycline is synthesized. Proton NMR experiments of tetracycline, spin-labeled tetracycline, and the diamagnetic reduced form in DMSO-d6 are reported. The signals observed in the NMR spectra are all assigned. The NMR data revealed that the spin label is attached to the C-2 amide group on ring A of tetracycline. The spin-labeled tetracycline is also tested in vitro for antitumor activity and is found to be active against leukemia P338/ADR cell line and in melanoma LOX cell line.