Structural interpretation of mutations and SNPs using STRAP-NT

Visualization of residue positions in protein alignments and mapping onto suitable structural models is an important first step in the interpretation of mutations or polymorphisms in terms of protein function, interaction, and thermodynamic stability. Selecting and highlighting large numbers of residue positions in a protein structure can be time-consuming and tedious with currently available software. Previously, a series of tasks and analyses had to be performed one-by-one to map mutations onto 3D protein structures; STRAP-NT is an extension of STRAP that automates these tasks so that users can quickly and conveniently map mutations onto 3D protein structures. When the structure of the protein of interest is not yet available, a related protein can frequently be found in the structure databases. In this case the alignment of both proteins becomes the crucial part of the analysis. Therefore we embedded these program modules into the Java-based multiple sequence alignment program STRAP-NT. STRAP-NT can simultaneously map an arbitrary number of mutations denoted using either the nucleotide or amino acid sequence. When the designations of the mutations refer to genomic sites, STRAP-NT translates them into the corresponding amino acid positions, taking intron-exon boundaries into account. STRAP-NT tightly integrates a number of current protein structure viewers (currently PYMOL, RASMOL, JMOL, and VMD) with which mutations and polymorphisms can be directly displayed on the 3D protein structure model. STRAP-NT is available at the PDB site and at http://www.charite.de/bioinf/strap/ or http://strapjava.de.     

Substrate-bonded hyaluronic acid exhibits a size-dependent stimulation of chondrogenic differentiation of stage 24 limb mesenchymal cells in culture

Extracellular matrix molecules including glycosaminoglycans have been implicated in several differentiative and morphogenetic processes including cell aggregation and migration. Previous reports have shown that plating of stage 24 limb mesenchyme cells onto hyaluronic acid (HA) bonded to the culture substrate causes an increase in the number of cells exhibiting chondrogenesis. This increased chondrogenesis is now shown to be dependent upon the source of the HA. When limb mesenchymal cells are plated onto HA from bovine vitreous humor, human umbilical cord, or large molecular weight HA (Healon), increased chondrogenesis is observed only on the bovine vitreous humor HA. Unsulfated chondroitin, which has a structure and charge density similar to those of HA, is capable of enhancing chondrogenesis, while cells plated onto sulfated glycosaminoglycan substrates are indistinguishable from controls. The evidence in this report suggests that the differentiation response is related to the molecular size of the HA bound to the culture substrate. Healon and human umbilical cord HA are ineffective because their molecular weight is too large, while smaller HA derived from these larger molecules or normally present in bovine vitreous humor preparations stimulates the chondrogenic differentiation of stage 24 limb mesenchymal cells in culture. The most active size class of HA elutes from a Sepharose CL-2B column with a Kav between 0.6 and 0.7 and, thus, has a molecular weight of approximately 200,000-400,000. These observations reinforce the hypothesis that local cues have an informational effect on the differentiation of chick limb mesenchymal cells.     

Electron transfer mediator micro-biosensor fabrication by organic plasma process

We propose a new strategy for constructing a mediator-type biosensor as a Bio-MicroElectroMechanical Systems (BioMEMS) application. A vinylferrocene plasma-polymerized film (PPF) was deposited directly onto the surface of an electrode under dry conditions. The resulting redox film was extremely thin, adhered well onto a substrate (electrode), and had a highly crosslinked network structure. This technique, capable of polymeric deposition of any kind of monomer, can also serve the purpose of anti-fouling coating, or layer-to-layer interface creation. With a subsequent plasma process, additional polymeric layer of hydrophilic acetonitrile was superimposed onto the existing vinylferrocene-PPF surface to offer crucial features such that the wettability could be adjusted for a better electron transfer, and amino functional groups could be attached to immobilize a large amount of enzyme. Based upon this scheme, the device fabrication could be designed in a manner that the whole procedure was made up of dry wafer-handling processes, which is compatible with mass production. A prototype device was fabricated to have an array of needle-shaped amperometric micro-biosensors. The resultant thin polymer layer carried a large number of the mediator molecules, accomplishing a lower overpotential (+410 mV) and a rapid response time (<5s). Stressing the advantages of the plasma polymerization process together with some additional features accomplished in our device fabrication, we would discuss new possibilities in the field of BioMEMS.     

A radiographic technique of interest to physical anthropologists

A radiographic technique for processing a large number of human crania was developed to aid in the diagnosis of porotic hyperostosis in a large skeletal population. These images are made directly onto photographic paper, thereby reducing costs and increasing the rate of processing. The technique is especially well suited for radiographing human skeletal material and gives excellent diagnostic image quality.     

Thermodynamic properties of the kinesin neck-region docking to the catalytic core

Kinesin motors move on microtubules by a mechanism that involves a large, ATP-triggered conformational change in which a mechanical element called the neck linker docks onto the catalytic core, making contacts with the core throughout its length. Here, we investigate the thermodynamic properties of this conformational change using electron paramagnetic resonance (EPR) spectroscopy. We placed spin probes at several locations on the human kinesin neck linker and recorded EPR spectra in the presence of microtubules and either 5'-adenylylimidodiphosphate (AMPPNP) or ADP at temperatures of 4-30 degrees C. The free-energy change (DeltaG) associated with AMPPNP-induced docking of the neck linker onto the catalytic core is favorable but small, about 3 kJ/mol. In contrast, the favorable enthalpy change (DeltaH) and unfavorable entropy change (TDeltaS) are quite large, about 50 kJ/mol. A mutation in the neck linker, V331A/N332A, results in an unfavorable DeltaG for AMPPNP-induced zipping of the neck linker onto the core and causes motility defects. These results suggest that the kinesin neck linker folds onto the core from a more unstructured state, thereby paying a large entropic cost and gaining a large amount of enthalpy.     

双色荧光杂交芯片在近交系小鼠遗传监测中的应用

应用一种新的高通量SNP检测方法-双色荧光杂交芯片技术进行近交系小鼠遗传监测。应用双色荧光杂交芯片技术对4个品系近交系小鼠的多个基因组DNA样本进行SNP分型,整合6个SNP位点的芯片杂交信息,对样本所属品系进行判断。研究结果表明SNP检测方法-双色荧光杂交芯片技术能够对选定的6个SNP位点进行高准确率分型;双色荧光杂交芯片技术是一种高通量SNP检测的良好工具,适合于对少量近交系品系来源的大样本量小鼠进行遗传污染监测和品系鉴定,并具有扩大应用的潜力。     

Integrated analysis of gene expression and copy number data on gene shaving using independent component analysis

DNA microarray gene expression and microarray-based comparative genomic hybridization (aCGH) have been widely used for biomedical discovery. Because of the large number of genes and the complex nature of biological networks, various analysis methods have been proposed. One such method is "gene shaving," a procedure which identifies subsets of the genes with coherent expression patterns and large variation across samples. Since combining genomic information from multiple sources can improve classification and prediction of diseases, in this paper we proposed a new method, "ICA gene shaving" (ICA, independent component analysis), for jointly analyzing gene expression and copy number data. First we used ICA to analyze joint measurements, gene expression and copy number, of a biological system and project the data onto statistically independent biological processes. Next, we used these results to identify patterns of variation in the data and then applied an iterative shaving method. We investigated the properties of our proposed method by analyzing both simulated and real data. We demonstrated that the robustness of our method to noise using simulated data. Using breast cancer data, we showed that our method is superior to the Generalized Singular Value Decomposition (GSVD) gene shaving method for identifying genes associated with breast cancer.     

Immunocytochemical detection of anti-ACTH reactivity in pancreatic islet cells of normal and steroid diabetic rats

Morphologically different pancreatic islet cells of the rat reveal immunoreactivity under incubation with anti-ACTH 1-39. In normal rats, anti-ACTH reactivity is detectable predominantly in peripheral, polygonal cells. Paraffin serial section treated alternately with anti-ACTH and anti-glucagon show that the major portion of the anti-ACTH reactivity is localized in A cells. A few A cells, however, lack anti-ACTH reactivity. Anti-ACTH reactivity was also detected in individuals, round-to-oval, occasionally quite large and more centrally situated cells of pancreatic islets. In contrast, islets of steroid diabetic rats reveal a different distribution islets. In contrast, islets of steroid diabetic rats reveal a different distribution of anti-ACTH reactive cells. The number of peripheral reacting cells is greatly reduced; whereas there is an increase in the number of immunoreactive, large, round or polygonal cells, which are distributed throughout the islet. Preliminary investigations indicate that adsorption of ACTH 18-39 and glucagon onto anti-ACTH 1-39 reduces immunoreactivity in normal and steroid diabetic rats, whereas adsorption of ACTH 1-24 does not.     

Protein-protein recognition analyzed by docking simulation.

Antibody-lysozyme and protease-inhibitor complexes are reconstituted by docking lysozyme as a rigid body onto the combining site of the antibodies and the inhibitors onto the active site of the proteases. Simplified protein models with one sphere per residue are subjected to simulated annealing using a crude energy function where the attractive component is proportional to the interface area. The procedure finds clusters of orientations in which a steric fit between the two protein components is achieved over a large contact surface. With five out of six complexes, the native structure of the complexes determined by X-ray crystallography is among those retained. Docked complexes are then subjected to conformational energy refinement with full atomic detail. With Fab HyHEL 5 and lysozyme, a native-like complex has the lowest refined energy. It can also be retrieved when starting with the X-ray structure of free lysozyme. However, some non-native complexes cannot be rejected: they form large interfaces, have a large number of H-bonds, and few unpaired polar groups. While these are necessary features of protein-protein recognition, they are not sufficient in determining specificity.     

Immunocytochemical detection of anti-ACTH reactivity in pancreatic islet cells of normal and steroid diabetic rats

Summary Morphologically different pancreatic islet cells of the rat reveal immunoreactivity under incubation with anti-ACTH 1-39. In normal rats, anti-ACTH reactivity is detectable predominantly in peripheral, polygonal cells. Paraffin serial section treated alternately with anti-ACTH and antiglucagon show that the major portion of the anti-ACTH reactivity is localized in A cells. A few A cells, however, lack anti-ACTH reactivity. Anti-ACTH reactivity was also detected in individual, round-to-oval, occasionally quite large and more centrally situated cells of pancreatic islets. In contrast, islets of steroid diabetic rats reveal a different distribution of anti-ACTH reactive cells. The number of peripheral reacting cells is greatly reduced; whereas there is an increase in the number of immunoreactive, large, round or polygonal cells, which are distributed throughout the islet. Preliminary investigations indicate that adsorption of ACTH 18-39 and glucagon onto anti-ACTH 1-39 reduces immunoreactivity in normal and steroid diabetic rats, whereas adsorption of ACTH 1-24 does not.This study was supported by the Deutsche Forschungsgemeinschaft Kl 426/2     

Enhanced Growth of Endothelial Precursor Cells on PCG-Matrix Facilitates Accelerated,Fibrosis-Free,Wound Healing: A Diabetic Mouse Model

Diabetes mellitus (DM)-induced endothelial progenitor cell (EPC) dysfunction causes impaired wound healing, which can be rescued by delivery of large numbers of ‘normal’ EPCs onto such wounds. The principal challenges herein are (a) the high number of EPCs required and (b) their sustained delivery onto the wounds. Most of the currently available scaffolds either serve as passive devices for cellular delivery or allow adherence and proliferation, but not both. This clearly indicates that matrices possessing both attributes are ‘the need of the day’ for efficient healing of diabetic wounds. Therefore, we developed a system that not only allows selective enrichment and expansion of EPCs, but also efficiently delivers them onto the wounds. Murine bone marrow-derived mononuclear cells (MNCs) were seeded onto a PolyCaprolactone-Gelatin (PCG) nano-fiber matrix that offers a combined advantage of strength, biocompatibility wettability; and cultured them in EGM2 to allow EPC growth. The efficacy of the PCG matrix in supporting the EPC growth and delivery was assessed by various in vitro parameters. Its efficacy in diabetic wound healing was assessed by a topical application of the PCG-EPCs onto diabetic wounds. The PCG matrix promoted a high-level attachment of EPCs and enhanced their growth, colony formation, and proliferation without compromising their viability as compared to Poly L-lactic acid (PLLA) and Vitronectin (VN), the matrix and non-matrix controls respectively. The PCG-matrix also allowed a sustained chemotactic migration of EPCs in vitro. The matrix-effected sustained delivery of EPCs onto the diabetic wounds resulted in an enhanced fibrosis-free wound healing as compared to the controls. Our data, thus, highlight the novel therapeutic potential of PCG-EPCs as a combined ‘growth and delivery system’ to achieve an accelerated fibrosis-free healing of dermal lesions, including diabetic wounds.     

A method for application of samples to matrix-assisted laser desorption ionization time-of-flight targets that enhances peptide detection

Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry has become a fundamental tool for the identification and analysis of peptides and proteins. MALDI-TOF is well suited for the analysis of complex biological mixtures because samples are crystallized onto a solid support that can be washed to remove contaminants and salts prior to laser desorption. A number of approaches for immobilizing samples onto MALDI targets have been put forth. These include the use of different chemical matrices and the immobilization of samples onto different solid supports. In large part though, the preparation of MALDI targets has been an empirical exercise that often requires a unique series of conditions for every sample. Here, a simple method for the application of peptide mixtures onto MALDI targets is put forth. This method differs because peptides are added directly to a sample of nitrocellulose dissolved in acetone, allowing them to interact in solution-phase organic solvent. This solution-phase mixture is then spotted to the MALDI target and evaporated, forming a homogenous solid surface for laser desorption. This procedure is robust, highly sensitive, tolerant to detergents, and easily learned. In our hands, the method provides as much as a 10-fold enhancement to the detection of tryptic peptide fragments derived from in-gel digests.     

Blind source separation and the analysis of microarray data.

We develop an approach for the exploratory analysis of gene expression data, based upon blind source separation techniques. This approach exploits higher-order statistics to identify a linear model for (logarithms of) expression profiles, described as linear combinations of "independent sources." As a result, it yields "elementary expression patterns" (the "sources"), which may be interpreted as potential regulation pathways. Further analysis of the so-obtained sources show that they are generally characterized by a small number of specific coexpressed or antiexpressed genes. In addition, the projections of the expression profiles onto the estimated sources often provides significant clustering of conditions. The algorithm relies on a large number of runs of "independent component analysis" with random initializations, followed by a search of "consensus sources." It then provides estimates for independent sources, together with an assessment of their robustness. The results obtained on two datasets (namely, breast cancer data and Bacillus subtilis sulfur metabolism data) show that some of the obtained gene families correspond to well known families of coregulated genes, which validates the proposed approach.     

Adsorption of LLCMK2 cell-grown Sendai virus onto human red blood cells and its release from the virus adsorbed cells

An early stage of virus adsorption was studied in a system of Sendai virus metabolically labeled with [3H]leucine in LLCMK2 cells and of human red blood cells (RBCs). The efficiency of viral release from the virus-bound RBCs by incubation at 37 C depended on the number of virus particles which had been used for adsorption onto the RBC at 4 C. When 7.8 x 10(2) virus particles were previously adsorbed onto the RBC at 4 C, most of the viruses were dissociated from the RBC at 37 C. In the case of adsorption of 3 to 12 virus particles per RBC, however, most of the viruses were not dissociated from the RBC by incubation at 37 C. Such RBC-bound viruses were released by incubation with various bacterial neuraminidases (Clostridium perfringens, etc.) or with a large number of LLCMK2 cell-grown Sendai virus (LLCMK2-Sendai) particles, but not released by treatment with hemagglutinin-neuraminidase protein (Sendai-gp) isolated from egg-grown Sendai virus.     

Rapid and automated enumeration of viable bacteria in compost using a micro-colony auto counting system

The micro-colony method was used to enumerate viable bacteria in composts. Cells were vacuum-filtered onto polycarbonate filters and incubated for 18 h on LB medium at 37 degrees C. Bacteria on the filters were stained with SYBR Green II, and enumerated using a newly developed micro-colony auto counting system which can automatically count micro-colonies on half the area of the filter within 90 s. A large number of bacteria in samples retained physiological activity and formed micro-colonies within 18 h, whereas most could not form large colonies on conventional media within 1 week. The results showed that this convenient technique can enumerate viable bacteria in compost rapidly for its efficient quality control.     

Influence of the conditions of trypsin immobilization onto Spherosil on coupling efficiency

Summary The effect of several parameters (pH, time of reaction, temperature, enzyme concentration) on trypsin immobilization onto glutaraldehyde-activated amine-Spherosil was investigated. This activated support could be stored over long periods of time without any important loss of capacity for trypsin coupling. When increasing the amount of trypsin bound to the carrier, enzymatic activity shows an optimal value, beyond which an augmentation of Spherosil enzyme content results in a lowered activity. The influence of the number of available reactive aldehyde groups on silica was investigated by coupling L-lysine to activated support either prior to or simulataneously with trypsin immobilization. In both cases, the activity of trypsin derivatives is decreased when L-lysine concentration is increased, yet the activity of trypsin derivatives is never equal to zero, even in presence of a large excess of L-lysine. This suggests the presence of two types of reactive groups on the activated support.     

Hominid divergence and speech evolution

Hominids evolved from a population which diverged from other hominoids during the Mio-Pliocene. This population was perhaps forced by ecological conditions and competitive exclusion to rely more on tools, gathering, hunting, vocal communication and memory, under whose mutually positively reinforcing effects the hominids diverged. The ape ancestors may have been forced into the forests (or they may have forced hominids onto the savanna), while hominids adapted to a plains, hunting econiche.Speech was selected for because verbal symbols served as retrieval cues for a large number of complex concepts and were transmissible, and thus could be used to influence food-getting and other behavior by the social group.Speech is dependent on three inherited entities: (1) anatomical and neurological adaptations which allow vocalization of a wide range of phonemes in rapid succession and which allow for (2) duality of patterning, thereby promoting a large number of words, and (3) encoding, which greatly increases the rate at which verbal information transfer can occur. Speech may have evolved through small hominid groups using progressively more phonemes in an increasingly blended manner, with encoding subsequently being selected for. Neandertals apparently could not encode speech and could speak only a restricted range of phonemes. Their expanded cranial capacity may have been selected for to store ambiguous and slowly transmitted verbal data.     

Generating tandem repeats by cloning with double initiator fragments

The ability to generate tandem repeats of a DNA sequence has proven important for a large variety of studies of DNA structure and function. The most commonly used method to produce tandem repeats involves cloning of an oligomerized monomer sequence that contains asymmetric overlapping ends, but, in practice, this approach is inefficient because of the circularization of oligomers before they ligate into vector. Described here is a method that circumvents this problem by the use of two separate oligomerization reactions, each containing an initiator fragment onto which monomer polymerizes without circularization. Subsequent mixing of the two reactions permits circularization, generating a viable plasmid containing the sum of the added repeats from each reaction. A variation of this method is also demonstrated that permits the synthesis of constructs with a defined number of repeats.     

Calcitonin receptors of human osteoclastoma

Osteoclast-rich cultures were prepared by disaggregation of osteoclastomas (giant cell tumour of bone) and settlement onto glass or plastic surfaces. Autoradiography using [125I]-salmon calcitonin ([125I]-sCT) revealed specific binding only to multinucleate giant cells (osteoclasts) and a minor population of mononuclear cells. [125I]-sCT competitive binding studies indicated a Kd of 5 x 10(-10) M and receptor number of approximately 1 million sites/osteoclast. sCT treatment resulted in a dose-dependent rise in cAMP (EC50 10(-10) M). Homogenates of an osteoclastoma also demonstrated specific binding of [125I]-sCT. Chemical cross-linking of a labelled synthetic sCT derivative. [125I]-[Arg11,18,Lys14]-sCT, using disuccinimidyl suberate, resulted in labelling of a receptor component of approximate Mr 85-90,000. The multinucleate giant cells (osteoclasts) of human osteoclastomas possess large number of CT receptors which exhibit the same binding kinetics and apparent Mr as those of other CT target cells.