Binding of Ions to Oligopeptides

A model for the binding of ions to oligopeptides, in which nearest neighbor interactions are considered is developed. Equations for the titration curves are derived The apparent association constants are determined as a function of the degree of polymerization and of the interactions between nearest neighbors.     

Supported phospholipid/alkanethiol biomimetic membranes: insulating properties.

A novel model lipid bilayer membrane is prepared by the addition of phospholipid vesicles to alkanethiol monolayers on gold. This supported hybrid bilayer membrane is rugged, easily and reproducibly prepared in the absence of organic solvent, and is stable for very long periods of time. We have characterized the insulating characteristics of this membrane by examining the rate of electron transfer and by impedance spectroscopy. Supported hybrid bilayers formed from phospholipids and alkanethiols are pinhole-free and demonstrate measured values of conductivity and resistivity which are within an order of magnitude of that reported for black lipid membranes. Capacitance values suggest a dielectric constant of 2.7 for phospholipid membranes in the absence of organic solvent. The protein toxin, melittin, destroys the insulating capability of the phospholipid layer without significantly altering the bilayer structure. This model membrane will allow the assessment of the effect of lipid membrane perturbants on the insulating properties of natural lipid membranes.     

Spontaneous development of anti-hepatitis B virus envelope (anti-idiotypic) antibodies in animals immunized with human liver endonexin II or with the F(ab')2 fragment of anti-human liver endonexin II immunoglobulin G: evidence for a receptor-ligand-like relationship between small hepatitis B surface antigen and endonexin II.

In a previous study, we have identified endonexin II (E-II) on human liver plasma membranes as a specific, Ca(2+)-dependent, small hepatitis B surface antigen (HBsAg)-binding protein. In this article, we describe the spontaneous development of anti-HBs antibodies in rabbits immunized with native or recombinant human liver E-II and in chickens immunized with the F(ab')2 fragment of rabbit anti-human liver E-II immunoglobulin G. Anti-HBs activity was not observed in rabbits immunized with rat liver E-II. Cross-reactivity of anti-E-II antibodies to HBsAg epitopes was excluded, since anti-HBs and anti-E-II activities can be separated by E-II affinity chromatography. The existence of an anti-idiotypic antibody is further demonstrated by competitive binding of human liver E-II and this antibody (Ab2) to small HBsAg, suggesting that Ab2 mimics a specific E-II epitope that interacts with small HBsAg. In addition, it was demonstrated that anti-HBs antibodies developed in rabbits after immunization with intact human liver E-II or in chickens after immunization with F(ab')2 fragments of rabbit anti-human liver E-II immunoglobulin G recognize the same epitopes on small HBsAg. These findings strongly indicate that human liver E-II is a very specific small HBsAg-binding protein and support the assumption that human liver E-II is the hepatitis B virus receptor protein.     

Correlation and prediction of gene expression level from amino acid and dipeptide composition of its protein

Background  

A large number of papers have been published on analysis of microarray data with particular emphasis on normalization of data, detection of differentially expressed genes, clustering of genes and regulatory network. On other hand there are only few studies on relation between expression level and composition of nucleotide/protein sequence, using expression data. There is a need to understand why particular genes/proteins express more in particular conditions. In this study, we analyze 3468 genes of Saccharomyces cerevisiae obtained from Holstege et al., (1998) to understand the relationship between expression level and amino acid composition.     

Determination and occurrence of polybrominated diphenyl ethers in maternal adipose tissue from inhabitants of Singapore

Polybrominated diphenyl ethers (PBDEs), as a specific group of brominated flame retardants (BFR), are used in a variety of consumer products including electronics and household furnishings. In recent years, a marked increase in the levels of PBDEs in human biological tissues and fluids, especially breast milk, has been reported in several countries. However, few data are available from countries in the Asia-pacific region, including Singapore. This study presents a validated method procedure and the first available data of the concentrations of PBDE congeners: PBDE-47 (2,2,4,4-Tetrabromodiphenyl ether), PBDE-99 (2,2',4,4',5-Pentabromodiphenyl ether), PBDE-100 (2,2',4,4',6-Pentabromodiphenyl ether), PBDE-153 (2,2',4,4',5,5'-Hexabromodiphenyl ether), PBDE-154 (2,2',4,4',5,6'-Hexabromodiphenyl ether) in maternal adipose tissue collected from inhabitants of Singapore. Microwave-assisted extraction (MAE) of PBDEs spiked adipose tissues coupled with GC-MS analysis achieved comparable recoveries to a conventional Soxhlet Extraction (SE) procedure of between 70 and 130%. MAE also yielded comparable precision data (variance less than 13%) relative to the SE procedure. Spiked Carbon-13 PBDE congeners were also used as surrogates for MAE quality assurance and confirmed the efficiency of the procedure. PBDE congeners were detected in all of 16 maternal adipose tissues collected in Singapore, where levels were comparable to available data from Belgium.     

Determination of flavonoids from Orthosiphon stamineus in plasma using a simple HPLC method with ultraviolet detection

A simple liquid chromatographic method was developed for the simultaneous determination of flavonoids from Orthosiphon stamineus Benth, namely sinensitin, eupatorin and 3'-hydroxy-5,6,7,4'-tetramethoxyflavone, in plasma. Prior to analysis, the flavonoids and the internal standard (naproxen) were extracted from plasma samples using a 1:1 mixture of ethyl acetate and chloroform. The detection and quantification limits for the three flavonoids were similar being 3 and 5 ng/ml, respectively. The within-day and between-day accuracy values, expressed as percentage of true values, for the three flavonoids were between 95 and 107%, while the corresponding precision, expressed as coefficients of variation, for the three flavonoids were less than 14%. In addition, the mean recovery values of the extraction procedure for all the flavonoids were between 92 and 114%. The calibration curves were linear over a concentration range of 5-4000 ng/ml. The present method was applied to analyse plasma samples obtained from a pilot study using rats in which the mean absolute oral bioavailability values for sinensitin, eupatorin and 3'-hydroxy-5,6,7,4'-tetramethoxyflavone was 9.4, 1.0 and 1.5%, respectively.     

Maximizing interferon-gamma production by Chinese hamster ovary cells through temperature shift optimization: experimental and modeling

The Chinese hamster ovary (CHO) cell line producing interferon-gamma (IFN-gamma) exhibits a 2-fold increase in specific productivity when grown at 32 degrees C compared to 37 degrees C. Low temperature also causes growth arrest, meaning that the cell density is significantly lower at 32 degrees C, nutrients are consumed at a slower rate and the batch culture can be run for a longer period of time prior to the onset of cell death. At the end of the batch, product concentration is doubled at the low temperature. However, the batch time is nearly doubled as well, and this causes volumetric productivity to only marginally improve by using low temperature. One approach to alleviate the problem of slow growth at low temperature is to utilize a biphasic process, wherein cells are cultured at 37 degrees C for a period of time in order to obtain reasonably high cell density and then the temperature is shifted to 32 degrees C to achieve high specific productivity. Using this approach, it is hypothesized that IFN-gamma volumetric productivity would be maximized. We developed and validated a model for predicting the optimal point in time at which to shift the culture temperature from 37 degrees C to 32 degrees C. It was found that by shifting the temperature after 3 days of growth, the IFN-gamma volumetric productivity is increased by 40% compared to growth and production at 32 degrees C and by 90% compared to 37 degrees C, without any decrease in total production relative to culturing at 32 degrees C alone. The modeling framework presented here is applicable for optimizing controlled proliferation processes in general.     

Synthesis, structural studies and solubility properties of zinc(II), nickel(II) and copper(II) complexes of bulky tris(triazolyl)borate ligands

Tris(triazolyl)borate (Ttz) ligands are sterically similar to tris(pyrazolyl)borate (Tp) but complexes of Ttz show improved solubility in water and alcohols due to their propensity for forming hydrogen bonds. Recently developed bulky tris(triazolyl)borate ligands can produce four and five coordinate transition metal complexes and serve as models for enzyme active sites in an aqueous environment. Herein we report the synthesis of such complexes, i.e. (Ttz^tBu,Me)ZnCl, (Ttz^tBu,Me)ZnBr, (Ttz^tBu,Me)NiCl, and (Ttz^tBu,Me)CuCl, which were analyzed by X-ray crystallographic and spectroscopic methods [Ttz^tBu,Me = tris(3-t-butyl-5-methyl-1,2,4-triazolyl)borate]. (Ttz^tBu,Me)ZnCl crystallizes as two different polymorphs with cubic and monoclinic symmetry. Both polymorphs of (Ttz^tBu,Me)ZnCl and (Ttz^tBu,Me)ZnBr have tetrahedral zinc atoms whereas the geometries at the metal in (Ttz^tBu,Me)NiCl and (Ttz^tBu,Me)CuCl are distorted tetrahedral. All complexes are methanol soluble and they also dissolve in methanol/water mixtures with up to 60% water.