Structural evolution and strain induced mixing in Cu-Co composites studied by transmission electron microscopy and atom probe tomography.

A Cu-Co composite material is chosen as a model system to study structural evolution and phase formations during severe plastic deformation. The evolving microstructures as a function of the applied strain were characterized at the micro-, nano-, and atomic scale-levels by combining scanning electron microscopy and transmission electron microscopy including energy-filtered transmission electron microscopy and electron energy-loss spectroscopy. The amount of intermixing between the two phases at different strains was examined at the atomic scale using atom probe tomography as complimentary method. It is shown that Co particles are dissolved in the Cu matrix during severe plastic deformation to a remarkable extent and their size, number, and volume fraction were quantitatively determined during the deformation process. From the results, it can be concluded that supersaturated solid solutions up to 26 at.% Co in a fcc Cu-26 at.% Co alloy are obtained during deformation. However, the distribution of Co was found to be inhomogeneous even at the highest degree of investigated strain.

Low-energy electron scattering in carbon-based materials analyzed by scanning transmission electron microscopy and its application to sample thickness determination.

High-angle annular dark-field scanning transmission electron microscopy (HAADF STEM) at low energies (≤30 keV) was used to study quantitatively electron scattering in amorphous carbon and carbon-based materials. Experimental HAADF STEM intensities from samples with well-known composition and thickness are compared with results of Monte Carlo simulations and semiempirical equations describing multiple electron scattering. A well-defined relationship is found between the maximum HAADF STEM intensity and sample thickness which is exploited (a) to derive a quantitative description for the mean quadratic scattering angle and (b) to calculate the transmitted HAADF STEM intensity as a function of the relevant materials parameters and electron energy. The formalism can be also applied to determine TEM sample thicknesses by minimizing the contrast of the sample as a function of the electron energy.

Integrin affinity modulation.

Integrin cell-adhesion receptors mediate interactions between cells and the extracellular matrix. Dynamic regulation of integrin adhesive function is termed 'activation' or 'inside-out' signalling. Activation is key to integrin function in processes as diverse as cell migration, the organization of the extracellular matrix and platelet aggregation. Consequently, there has been an intense effort to elucidate the molecular mechanism of integrin activation. This has resulted in the recent identification of novel cytoplasmic partners for integrins and the emerging characterization of the signal-transduction pathways that regulate integrin 'inside-out' signalling. Here, the authors review the recent developments that have provided us with an increased understanding of the basis of integrin activation.

Beta 1 integrin-dependent and -independent polymerization of fibronectin.

The mouse cell line GD25, which lacks expression of the beta 1 family of integrin heterodimers due to disruption of the beta 1 integrin subunit gene, was used for expression of full-length cDNA coding for splice variant A of the mouse beta 1 integrin subunit. In a stably transformed clone (GD25-beta 1A), the expressed protein was found to form functional heterodimeric receptors together with the subunits alpha 3, alpha 5, and alpha 6. Both GD25 and GD25-beta 1A attached to fibronectin and formed focal contacts which contained alpha v beta 3, but no detectable alpha 5 beta 1A. The presence of GRGDS peptide allowed alpha 5 beta 1A to locate to focal contacts of GD25-beta 1A cultured on fibronectin, while the beta 1-null GD25 cells were unable to attach under these conditions. Affinity chromatography revealed that alpha 5 beta 1A and alpha v beta 3 could bind to a large cell-binding fragment of fibronectin. alpha 5 beta 1A strongly promoted polymerization of fibronectin into a fibrillar network on top of the cells. Whereas little alpha v beta 3 was colocalized with the fibronectin fibrils in GD25-beta 1A cells, this integrin was able to support fibronectin fibril polymerization in GD25 cells. However, the alpha v beta 3-induced polymerization was less efficient and occurred mainly in dense cultures of the GD25 cells. Thus, while both alpha 5 beta 1A and alpha v beta 3 are able to support adhesion to fibronectin, alpha v beta 3 dominates in the formation of focal contacts, and alpha 5 beta 1A has a prime function in fibronectin matrix assembly. This is the first report on fibronectin matrix assembly in the absence of beta 1 integrins.

Lack of beta 1 integrin gene in embryonic stem cells affects morphology, adhesion, and migration but not integration into the inner cell mass of blastocysts.

A gene trap-type targeting vector was designed to inactivate the beta 1 integrin gene in embryonic stem (ES) cells. Using this vector more than 50% of the ES cell clones acquired a disruption in the beta 1 integrin gene and a single clone was mutated in both alleles. The homozygous mutant did not produce beta 1 integrin mRNA or protein, while alpha 3, alpha 5, and alpha 6 integrin subunits were transcribed but not detectable on the cell surface. Heterozygous mutants showed reduced beta 1 expression and surface localization of alpha/beta 1 heterodimers. The alpha V subunit expression was not impaired on any of the mutants. Homozygous ES cell mutants lacked adhesiveness for laminin and fibronectin but not for vitronectin and showed a reduced association with a fibroblast feeder layer. Furthermore, they did not migrate towards chemoattractants in fibroblast medium. None of these functions were impaired in heterozygous mutants. Scanning electron microscopy revealed that homozygous cells showed fewer cell-cell junctions and had many microvilli not usually found on wild type and heterozygous cells. This profound change in cell shape is not associated with gross alterations in the expression and distribution of cytoskeletal components. Unexpectedly, microinjection into blastocysts demonstrated full integration of homozygous and heterozygous mutants into the inner cell mass. This will allow studies of the consequences of beta 1 integrin deficiency in several in vivo situations.

Human monoclonal antibody against a tissue polypeptide antigen-related protein from a patient with a signet-ring cell carcinoma of the stomach.

For a comparative study of the humoral immunity of patients with gastric signet-ring cell carcinoma, lymphocytes from spleen and lymph nodes were fused with the heteromyeloma SPM4-0. Immunoglobulin-producing clones were primarily tested in binding assays on autologous and allogeneic tumor cells and tissues. One of the resulting human monoclonal antibodies, designated 56/16 (IgM, lambda), was found to be suitable for a detailed biochemical characterization. Immunoblotting and comparative two-dimensional gel electrophoresis on cell and tissue extracts as well as on preparations of the cytoskeleton revealed that the main epitope is not an integral membrane molecule but a degradation product of cytokeratin 8, which is a main component of the tumor marker, tissue polypeptide antigen. The Mr 38,000/45,000 antigen could be identified in tumor and normal tissues, with highest expression in secretory cells and organs. Thus, the human monoclonal antibody 56/16 might represent an immune response in the patient against breakdown products of cytokeratin 8, which are released from the tumor cells during cell division, secretion, or cell death. A possible association of the antibody with the secretory activity of signet-ring carcinoma cells is discussed.

Casein kinase II accumulation in the nucleolus and its role in nucleolar phosphorylation.

A rabbit antiserum against highly purified casein kinase II from mouse tumor cells was used for immunolocalization of the enzyme in fixed, permeabilized mouse cells. Casein kinase II was highly accumulated in nucleoli compared to the extra-nucleolar space of the nucleus or to the cytoplasma. Casein kinase II samples highly purified from the cytoplasma, from the extra-nucleolar fraction of the nucleus or from nucleoli exhibited no differences with respect to structure and function. All samples originally had an alpha 2 beta 2 structure (alpha, 42 kDa; beta, 24 kDa) showing formation of the alpha'-chain (36 kDa) only in the late steps of purification. The isoelectric point of the alpha-chain of all three samples was pH 7.7 and that of the beta-chain was pH 6.4-6.6. Using ATP or GTP, all three casein kinase II samples gave the same results of maximum phosphorylation of purified nucleolar marker phosphoproteins pp105/C23, pp135 and B23, yielding pp135 as one of the most highly phosphorylated proteins with an incorporation of about 75 phosphate groups per molecule pp135. Studies on optimum conditions of phosphorylation of nucleolar phosphoproteins by casein kinase II revealed that each of the protein substrates individually responded to alterations of assay parameters such as pH, magnesium ion and sodium chloride concentrations indicating that predominantly individual structural criteria were responsible for optimum phosphorylation. The determination of the apparent Km of casein kinase II for purified nucleolar phosphoproteins yielded values of 0.15 microM (pp105/C23), 0.1 microM (pp135) and 1.0 microM (B23) identifying them as high-affinity substrates of casein kinase II.

Concurrence of aldosterone, androgen, and cortisol secretion in adrenal venous effluents.

Adrenal effluent concentrations of aldosterone were measured along with testosterone, androstenedione, and cortisol in 17 women with idiopathic hirsutism. In the basal state, aldosterone secretion (higher concentrations vs peripheral blood) was demonstrated in 14 out of 16 of the women, in contrast to 8 out of 16 who demonstrated cortisol gradients. Nine women received 0.25 mgm beta 1-24 ACTH im and serial adrenal venous blood samples were obtained over the next 30 minutes. Parallel 30-40-fold increments were noted in aldosterone, androstenedione, and cortisol; testosterone increased only 8-fold. These marked changes in adrenal effluents were not observed in simultaneously monitored peripheral blood. Minimal changes in these steroid concentrations were noted in adrenal and peripheral blood in 7 women who received 2,000 IU hCG. One woman received a pressor dose of angiotensin II, resulting in a marked increase in adrenal vein aldosterone and a simultaneous decrease in cortisol. Since adrenal androgen secretion parallels cortisol, quantification of adrenal androgen secretion rates can be achieved by sampling the adrenal effluent and relating the androgen gradient to that of cortisol at any given time. In contrast, aldosterone secretion is often independent of cortisol, and thus cannot be estimated by comparison of adrenal gradients. ACTH administration, however, invariably stimulated aldosterone secretion, enabling us to quantify the "ACTH-related aldosterone secretion rate" from a comparison of maximal adrenal gradients of aldosterone vs cortisol. In 6 women, "ACTH-related aldosterone" secretion rate averaged 40 mug/day, roughly 20% of the total daily aldosterone secretion rate.

Calpain cleavage of integrin beta cytoplasmic domains.

We showed previously that the calcium-dependent protease, calpain, cleaves the cytoplasmic domain of the integrin beta3 subunit. To investigate whether susceptibility to calpain is a common feature of all integrin beta subunits, and to map calpain cleavage sites in different integrin beta tails, we treated recombinant cytoplasmic domains of integrin beta1A, beta1D, beta2, beta3 and beta7 subunits with purified calpain in vitro. We found that the cytoplasmic domains of all these integrin chains were cleaved by calpain. HPLC followed by mass spectrometry was used to identify calpain cleavage sites. These sites were clustered in the C-terminal half of the integrin beta cytoplasmic domains in regions flanking the two NXXY motifs, suggesting the possibility that the structural framework provided by these motifs is recognized by calpain. We used the knowledge of these cleavage sites to develop cleavage site-specific antibodies and to demonstrate cleavage of the beta1A cytoplasmic domain in intact platelets stimulated with calcium ionophore or thrombin. Thus susceptibility to calpain cleavage is common to integrin beta subunits, can be induced in intact cells, and appears to favor regions surrounding two conserved NXXY motifs.

Cell adhesion and integrin binding to recombinant human fibrillin-1.

Fibrillin-1 is a major constituent of tissue microfibrils that occur in most connective tissues, either in close association with or independent of elastin. To test possible cell-adhesive functions of this protein, we used recombinant human fibrillin-1 polypeptides produced in a mammalian expression system in cell attachment and solid-phase integrin binding assays. Fibrillin-1 polypeptides containing the single RGD sequence located in the fourth 8-cysteine domain, mediated distinct cell adhesion of a variety of cell lines and bound to purified integrin alphaVbeta3. Integrins alphaIIbbeta3, alpha5beta1, alpha2beta1 and alpha1beta1 did not interact with any of the recombinant fibrillin-1 peptides. Our results indicate a novel role for fibrillin-1 in cellular interactions mediated via an RGD motif that is appropriately exposed for recognition by integrin alphaVbeta3.

Prediction of cardiovascular risk in hemodialysis patients by data mining.

OBJECTIVES: The objective of this work was to contribute to the development, validation and application of data mining methods for prediction in decision support systems in medicine. The particular focus was on the prediction of cardiovascular risk factors in hemodialysis patients, specifically the interventricular septum (IVS) thickness of the heart of individual patients as an important quantitative indicator to diagnose left ventricular hypertrophy. The work was based on data from 63 long-term hemodialysis patients of the KfH Dialysis Centre in Jena, Germany. METHODS: The approach applied is based on data mining methods and involves four major steps: data based clustering, cluster based rule extraction, rulebase construction and cluster and rule based prediction. The methods employed include crisp and fuzzy algorithms. At each step, logical and medical validation of results was carried out. Different sets of randomly selected patient data were used to train, test and optimize the clusterbases and rulebases for prediction. RESULTS: Using the best clusterbase/rulebase combination designed, the IVS thickness cluster ('small' or 'large') was predicted correctly for 30 of the 35 patients with known IVS values in the training data set; no patient was predicted incorrectly and 5 were parity predicted. For the test data set, 4 of the 6 patients with known IVS values were predicted correctly, no patient incorrectly and 2 parity. These results did not substantially differ from those obtained using the second best clusterbase/rulebase combination which was finally recommended for use based on further performance criteria. The prediction of the IVS thickness clusters of the 22 patients with unknown IVS values also yielded good results that were (and could only be) validated by a medical individual risk assessment of these patients. CONCLUSIONS: The approach applied proved successful for the cluster and rule based prediction of a quantitative variable, such as IVS thickness, for individual patients from other variables relevant to the problem. The results obtained demonstrate the high potential of the approach and the methods developed and validated to support decision-making in hemodialysis and other fields of medicine by individual risk prediction.

IgG monoclonal antibodies that inhibit osteoinductivity of human bone matrix-derived proteins (hBMP/NCP).

Monoclonal hBMP/NCP (human bone morphogenetic protein and associated noncollagenous proteins) antibodies of the IgG class were produced. In vitro, 12 of 19 hBMP/NCP antibodies showed functional inhibition of hBMP/NCP-induced chondroneogenesis in a neonatal muscle tissue assay. Inducing factors were characterized by their inhibiting antibodies with immunoblotting. Several peptide factors seem to be involved in the cascade of induced chondro- and osteogenesis.

[Simultaneous estimation of phenobarbitone and valproic acid in the plasma by high performance liquid chromatography (author's transl)]. / Dosage simultané du phénobarbital et de l'acide valproïque dans le plasma par chromatographie liquide haute performance.

The authors describe a method of simultaneous estimation of phenobarbitone and valproic acid in the blood. After extraction with ether, these substances were estimated by high performance liquid chromatography. They were separated on a mu Bondapack C18 column. The detection involved a colorimetric procedure based on variation in colour of a solution of bromocresol purple. The technic used is simple, its reproducibility is satisfactory (coefficients of variation of 7.6% for phenobarbitone and 2.3% for valproic acid). The sensitivity was 13.8 mumol/l for phenobarbitone and 11.1 mumol/l for valproic acid. Parallel estimations by gas chromatography led to comparable results. This method is of interest in the supervision of anticonvulsant treatments, especially in pediatrics.

Dynamics of amino acid metabolism of primary human liver cells in 3D bioreactors.

The kinetics of 18 amino acids, ammonia (NH3) and urea (UREA) in 18 liver cell bioreactor runs were analyzed and simulated by a two-compartment model consisting of a system of 42 differential equations. The model parameters, most of them representing enzymatic activities, were identified and their values discussed with respect to the different liver cell bioreactor performance levels. The nitrogen balance based model was used as a tool to quantify the variability of runs and to describe different kinetic patterns of the amino acid metabolism, in particular with respect to glutamate (GLU) and aspartate (ASP).

[Drug use: a life choice or health threat]. / Genuss: Lebenschance oder Lebensbeschränkung.

The term "enjoyment" is used to denote a pleasurable, utility-creating activity or an act of consumption, which, however, may often be accompanied by subsequent risks, health-care costs or reduction in the quality of life. If viewed from the vantage point of health policy, we may quote several examples of consumer behaviour of this type, ranging first and foremost from the consumption of alcohol and nicotine to the use of "hard" drugs. But certain sports activities or sexual behaviour that may prove damaging to other persons must also be mentioned in this context. All these forms of consumption often lead to direct or indirect consequences to health and thereby result in increased health spending amounting to several thousand million DM. The decline in quality of life resulting from the above-mentioned forms of behaviour could be avoided through health promotion and prevention or, at the very least, through measures of rehabilitation, above all, information, counselling, and education, which represent medium- or long-term measures to that effect. Financial incentives or disincentives, for example, via an increase in co-payment in case of illness caused by risky behaviour, have not been as effective as had been expected by many proponents. Product-specific levies, risk-related payments and other financial incentives and disincentives are generally also not as effective as desired. Health policy measures aimed at influencing behaviour via financial measures are particularly ineffective in case of persons addicted to particular types of consumables, especially to drugs: Information, counselling and education represent non-financial forms of influencing and guiding behaviour with a greater scope for success.

Differences in health care spending across countries: statistical evidence.

The empirical evidence available for OECD countries suggests that economic factors play a major role and that demographic factors play a minor role in explaining differences in health care spending across countries. When countries are grouped on the basis of their health care systems, some significant cross-country differences result: countries with higher transfer rates (a larger share of collective financing) are not generally characterized by higher health care expenditures, and conversely, countries with a larger share of private financing (including higher coinsurance rates) do not have lower expenditures. Rather, the opposite holds true. Similar conclusions apply to the share of public versus private production of health goods. Furthermore, the results do not support the claims of those critics of universal public insurance systems who consider the expansion of the coverage to be a major source of expenditure growth. These findings cast serious doubt on the claim that cost containment can be achieved via market reforms that rely heavily on direct consumer payments and cost sharing as instruments of financing. A comparative analysis of the historic record of the United States, Canada, and the Federal Republic of Germany generally supports these conclusions; it also suggests that a greater degree of public penetration offers a better chance for control of health spending, particularly in periods of austerity. There is a strong presumption that health care systems relying on some overall control of spending generally are more cost-effective than those relying more on decentralized mechanisms of control. Services are more equitably distributed in relation to health and payment for health services is far more progressive in the former type of system.