Spectral studies on aluminum ion binding to the ligands with phenolic group(s): implications for the differences between N- and C-terminal binding sites of human serum apotransferrin

Both the binding and releasing of ferric ions in C-, and N-terminal binding sites of human serum transferrin are different. To understand the difference here the interactions of aluminum with the ligands containing phenolic group(s), including 8-hydroxyquinoline, salicylic acid, N,N'-di(2-hydroxybenzyl)ethylenediamine-N,N'-diacetic acid, N,N'-ethylenebis[2-(o-hydroxyphenolic)glycine], and human serum apotransferrin, respectively, are investigated by using UV difference and fluorescence spectra methods in 0.1 M N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid at pH 7.4. Aluminum binding produces a UV difference peak near 235 nm that is characteristic of phenolic groups binding to aluminum. The peak at 235 nm has been used to determine conditional binding constants of log K(Al-HBED)=8.88+/-0.74 and log K(Al-EHPG)=9.38+/-0.03. However, the effects of aluminum binding on the fluorescence intensity of N,N'-ethylenebis[2-(o-hydroxyphenolic)glycine], salicylic acid and N,N'-di(2-hydroxybenzyl) ethylenediamine-N,N'-diacetic acid, 8-hydroxyquinoline are disparate, the former showing a decrease and the latter an increase. At pH 7.4, there is N cdots, three dots, centered H-O type intramolecular hydrogen bond in 8-hydroxyquinoline, N,N'-di(2-hydroxybenzyl) ethylenediamine-N,N'-diacetic acid and O cdots, three dots, centered H-O type intramolecular hydrogen bond in salicylic acid, N,N'-ethylenebis[2-(o-hydroxyphenolic)glycine]. The effects of salts on the fluorescence intensity of the ligands containing phenolic group(s) show that fluorescence emission increases with the breaking of an N cdots, three dots, centered H-O type intramolecular hydrogen bond and fluorescence emission decreases with the breaking of an O cdots, three dots, centered H-O type intramolecular hydrogen bond. Fluorescence titrations of apotransferrin and both forms of monoferric transferrin with aluminum indicated that there is O cdots, three dots, centered H-O type intramolecular hydrogen bonds for the phenolic groups of Tyr426 and Tyr517 in the C-terminal binding site. While N cdots, three dots, centered H-O type intramolecular hydrogen bonds are found for the phenolic groups of Tyr95 and Tyr188 in the N-terminal binding site.     

Skin graft survival in genetically identical cloned pigs

Nuclear transfer technology allows for the reprogramming of somatic cells, and the production of embryonic stem cells and animals that are genetically identical in terms of nuclear DNA to the parental somatic cell. It is assumed that these products of nuclear transfer technology will be immunologically compatible to each other in spite of the fact that there are data that show differences in the expression patterns and phenotypes between animals produced by nuclear transfer. We have produced a series of cloned pigs from embryonic fibroblasts. Microsatellite analysis was used to confirm that the clones were genetically identical. Skin transplants were performed to assess immunological reactivity. Skin transplants between genetically identical cloned pigs were accepted, whereas third party grafts were rejected. Histological analysis of the grafts showed edema and mononuclear cell infiltrates in the recipient's skin in rejected grafts and not in grafts that were accepted. Our data supports the notion that genetically identical cloned pigs are immunologically compatible.     

Death receptor activation-induced hepatocyte apoptosis and liver injury

The TNFalpha receptor super-family consists of several members sharing a sequence homology in a unique function domain, the death domain, which is located in the intracellular portion of the receptor. These so-called death receptors, including Fas, TNF-R1 and TRAIL-R1/TRAIL-R2, are expressed on hepatocytes. When stimulated by their ligands, FasL, TNFalpha or TRAIL, respectively, the death receptors can activate multiple death domain-initiated apoptosis programs, including both extrinsic and intrinsic pathways. A cascade of caspases is activated, which cleave proteins important for the cell structure and function. Activation of the intrinsic pathway also leads to mitochondrial release of several apoptotic proteins and mitochondrial dysfunction, which kill the cell through both caspase-dependent and caspase-independent mechanisms. Death receptor-induced hepatocyte apoptosis contributes to the development of a number of liver diseases, including viral hepatitis, inflammatory hepatitis, Wilson's disease, alcoholic liver disease, endotoxiemia-induced liver failure and ischemia/reperfusion-induced liver damage. This article comprehensively reviews the mechanisms of induction and regulation of death receptor-initiated apoptosis in hepatocytes, examines how these molecular events affect our understanding of the pathogenesis of these diseases and further discusses the potential therapeutic application of the knowledge. We hope we can provide a cohesive and integrated perspective on the many aspects of these complicated processes.     

Highly efficient conversion of lactate to pyruvate using whole cells of Acinetobacter sp

On an industrial scale, the production of pyruvate at a high concentration from the cheaper lactate substrate is a valuable process. To produce pyruvate from lactate by whole cells, various lactate-utilizing microorganisms were isolated from soil samples. Among them, strain WLIS, identified as Acinetobacter sp., was screened as a pyruvate producer. For the pyruvate preparation from lactate, the preparative conditions were optimized with whole cells of the strain. The cells cultivated in the medium containing 100 mM of l-lactate showed the highest biotransformation efficiency from lactate to pyruvate. The optimized dry-cell concentration, pH, and temperature of reaction were 6 g/L, pH 7.0-7.5, and 30 degrees C, respectively. The influences of ethylenediaminetetraacetic acid (EDTA) and aeration on a biotransformation reaction were carried out under the test conditions. Under the optimized reaction conditions, l-lactate at concentrations of 200 and 500 mM were almost totally stoichiometrically converted into pyruvate in 8 and 12 h, respectively. About 60% of 800 mM of l-lactate was transformed into pyruvate in 24 h. This reduced conversion rate is probably due to the high substrate inhibition in biotransformation.     

A statistical approach to computer processing of cryo-electron microscope images: virion classification and 3-D reconstruction

The scattering density of the virus is represented as a truncated weighted sum of orthonormal basis functions in spherical coordinates, where the angular dependence of each basis function has icosahedral symmetry. A statistical model of the image formation process is proposed and the maximum likelihood estimation method computed by an expectation-maximization algorithm is used to estimate the weights in the sum and thereby compute a 3-D reconstruction of the virus particle. If multiple types of virus particle are represented in the boxed images then multiple 3-D reconstructions are computed simultaneously without first requiring that the type of particle shown in each boxed image be determined. Examples of the procedure are described for viruses with known structure: (1). 3-D reconstruction of Flockhouse Virus from experimental images, (2). 3-D reconstruction of the capsid of Nudaurelia Omega Capensis Virus from synthetic images, and (3). 3-D reconstruction of both the capsid and the procapsid of Nudaurelia Omega Capensis Virus from a mixture of unclassified synthetic images.     

Sample preparation and imaging of erythrocyte cytoskeleton with the atomic force microscopy

A novel method for the covalent attachment of erythrocytes to glass microscope coverslips that can be used to image intact cells and the cytoplasmic side of the cell membrane with either solid or liquid mode atomic force microscopy (AFM) is described. The strong binding of cells to the glass surface is achieved by the interaction of cell membrane carbohydrates to lectin, which is bound to N-5-azido-2-nitrobenzoyloxysuccinimide (ANBNOS)-coated coverslips (1). The effectiveness of this method is compared with the other commonly used methods of immobilizing intact erythrocytes on glass coverslips for AFM observations. Experimental conditions of AFM imaging of biologic tissue are discussed, and typical topographies of the extracellular and the cytoplasmic surfaces of the plasma membrane in the dry state and in the liquid state are presented. Comparison of the spectrin network of cell age-separated erythrocytes has demonstrated significant loss in the network order in older erythrocytes. The changes are quantitatively described using the pixel height histogram and window size grain analysis.     

PCAS – a precomputed proteome annotation database resource

Background  

Many model proteomes or "complete" sets of proteins of given organisms are now publicly available. Much effort has been invested in computational annotation of those "draft" proteomes. Motif or domain based algorithms play a pivotal role in functional classification of proteins. Employing most available computational algorithms, mainly motif or domain recognition algorithms, we set up to develop an online proteome annotation system with integrated proteome annotation data to complement existing resources.     

The inhibitory effect of BSP-A1/-A2 on protein kinase C and tyrosine protein kinase

Bovine seminal plasma contains a group of similar proteins, namely BSP-A1, BSP-A2, BSP-A3, and BSP-30-kDa (collectively called BSP proteins), and they are secreted by the seminal vesicles. In our study, we purified the BSP-A1/-A2 through affinity chromatography and found for the first time that BSP-A1/-A2 can inhibit the activity of protein kinase C (PKC) and tyrosine protein kinase (TPK). The inhibition was dose dependent. When the PKC and TPK activities are expressed as the logarithm of percentage activity taking the activity in the absence of the BSP-A1/-A2 as 100%, there is a linear relationship between the their activities and the dose of BSP-A1/-A2.     

Endogenous assays of DNA methyltransferases: Evidence for differential activities of DNMT1, DNMT2, and DNMT3 in mammalian cells in vivo

While CpG methylation can be readily analyzed at the DNA sequence level in wild-type and mutant cells, the actual DNA (cytosine-5) methyltransferases (DNMTs) responsible for in vivo methylation on genomic DNA are less tractable. We used an antibody-based method to identify specific endogenous DNMTs (DNMT1, DNMT1b, DNMT2, DNMT3a, and DNMT3b) that stably and selectively bind to genomic DNA containing 5-aza-2'-deoxycytidine (aza-dC) in vivo. Selective binding to aza-dC-containing DNA suggests that the engaged DNMT is catalytically active in the cell. DNMT1b is a splice variant of the predominant maintenance activity DNMT1, while DNMT2 is a well-conserved protein with homologs in plants, yeast, Drosophila, humans, and mice. Despite the presence of motifs essential for transmethylation activity, catalytic activity of DNMT2 has never been reported. The data here suggest that DNMT2 is active in vivo when the endogenous genome is the target, both in human and mouse cell lines. We quantified relative global genomic activity of DNMT1, -2, -3a, and -3b in a mouse teratocarcinoma cell line. DNMT1 and -3b displayed the greatest in vivo binding avidity for aza-dC-containing genomic DNA in these cells. This study demonstrates that individual DNMTs can be tracked and that their binding to genomic DNA can be quantified in mammalian cells in vivo. The different DNMTs display a wide spectrum of genomic DNA-directed activity. The use of an antibody-based tracking method will allow specific DNMTs and their DNA targets to be recovered and analyzed in a physiological setting in chromatin.